Formulations of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid

ABSTRACT

A pharmaceutical composition comprising Compound 1, (3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid), and at least one excipient selected from: a filler, a disintegrant, a surfactant, a binder, and a lubricant, the composition being suitable for oral administration to a patient in need thereof to treat a CFTR mediated disease such as Cystic Fibrosis. Processes of preparing pharmaceutical compositions comprising Compound 1 are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/523,493 filed Jul. 26, 2019, which is now abandoned, which is aContinuation of U.S. patent application Ser. No. 15/655,354, filed Jul.20, 2017, which is now abandoned, which is a Continuation of U.S. patentapplication Ser. No. 15/093,582, filed Apr. 7, 2016, which is nowabandoned; which is a Continuation of U.S. patent application Ser. No.13/750,069, filed Jan. 25, 2013, which is now abandoned; which claimspriority to U.S. provisional patent application Ser. Nos. 61/590,479,filed Jan. 25, 2012; 61/651,218, filed May 24, 2012; 61/691,898, filedAug. 22, 2012; and 61/708,691, filed Oct. 2, 2012, the entire contentsof all applications are incorporated herein by reference in theirentirety.

TECHNICAL FIELD OF INVENTION

The invention relates to pharmaceutical compositions comprising3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Compound 1),methods for manufacturing such compositions and methods foradministering pharmaceutical compositions comprising same.

BACKGROUND

CFTR is a cAMP/ATP-mediated anion channel that is expressed in a varietyof cells types, including absorptive and secretory epithelia cells,where it regulates anion flux across the membrane, as well as theactivity of other ion channels and proteins. In epithelia cells, normalfunctioning of CFTR is critical for the maintenance of electrolytetransport throughout the body, including respiratory and digestivetissue. CFTR is composed of approximately 1480 amino acids that encode aprotein made up of a tandem repeat of transmembrane domains, eachcontaining six transmembrane helices and a nucleotide binding domain.The two transmembrane domains are linked by a large, polar, regulatory(R)-domain with multiple phosphorylation sites that regulate channelactivity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia lead to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhance mucus accumulation inthe lung and the accompanying microbial infections that ultimately causedeath in CF patients. In addition to respiratory disease, CF patientstypically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease-causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 1000disease-causing mutations in the CF gene have been identified asreported by the scientific and medical literature. The most prevalentmutation is a deletion of phenylalanine at position 508 of the CFTRamino acid sequence, and is commonly referred to as F508del-CFTR. Thismutation occurs in approximately 70 percent of the cases of cysticfibrosis and is associated with a severe disease. Other mutationsinclude the R117H and G551D.

The deletion of residue 508 in F508del-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of F508del-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toF508del-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl— channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue 508 inF508del-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective endoplasmic reticulum (ER) processingof ATP-binding cassette (ABC) transporters by the ER machinery, has beenshown to be the underlying basis not only for CF disease, but for a widerange of other isolated and inherited diseases. The two ways that the ERmachinery can malfunction is either by loss of coupling to ER export ofthe proteins leading to degradation, or by the ER accumulation of thesedefective/misfolded proteins [Aridor M, et al., Nature Med., 5(7), pp745-751 (1999); Shastry, B. S., et al., Neurochem. International, 43, pp1-7 (2003); Rutishauser, J., et al., Swiss Med Wkly, 132, pp 211-222(2002); Morello, J P et al., TIPS, 21, pp. 466- 469 (2000); Bross P., etal., Human Mut., 14, pp. 186-198 (1999)].

Compound 1 in salt form is disclosed in International PCT Publication WO2007056341 as a modulator of CFTR activity and thus as a usefultreatment for CFTR-mediated diseases such as cystic fibrosis. Compound 1Form I, which is substantially crystalline and salt-free, is disclosedin United States Published Patent Application US20090170905, filed Dec.4, 2008. Compound 1 Form II and Compound 1 HCl salt Form A are disclosedin United States Published Patent Application US20110263654, filed Apr.7, 2011. All applications are incorporated in their entirety byreference herein.

Compound 1, as part of a combination with ivacaftor(N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide),has been granted a Breakthrough Therapy Designation from the Food andDrug Administration (FDA) for the treatment of cystic fibrosis, one ofonly two such grants at the time of the filing of this application (theother being for ivacaftor). This demonstrates a significant unmet needfor the effective treatment of the cause of cystic fibrosis oversymptomatic treatments. Additionally, a common challenge for drugsapproved by the FDA is the occasional lack of drug availability forpatients in need thereof. Accordingly, a significant unmet need existsfor the presently disclosed Compound 1 formulations and processes forpreparing them in a continuous and controlled manner.

SUMMARY

The invention relates to pharmaceutical compositions, pharmaceuticalpreparations, and solid dosage forms comprising3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Compound 1)which has the structure below:

In one aspect, the invention provides a pharmaceutical compositioncomprising:

a. Compound 1;

b. a filler;

c. a disintegrant;

d. a surfactant;

e. a lubricant; and

f. a glidant or a binder.

In other embodiments, Compound 1 is in substantially one of itscrystalline solid forms. In one embodiment, Compound 1 is insubstantially crystalline Form I (Compound 1 Form I). In one embodiment,Compound 1 is in substantially crystalline Form II (Compound 1 Form II).In one embodiment, Compound 1 is in substantially crystalline HCl saltform (Compound 1 HCl Salt Form A). It is understood that the term“Compound 1,” as used throughout, includes, amongst other forms,including non-crystalline forms, the following solid state forms:Compound 1 Form I, Compound 1 Form II, and/or Compound 1 HCl Salt FormA.

In some embodiments, the pharmaceutical composition comprises 25 mg to400 mg. In some embodiments, the pharmaceutical composition comprises 25mg of Compound 1. In some embodiments, the pharmaceutical compositioncomprises 50 mg of Compound 1. In some embodiments, the pharmaceuticalcomposition comprises 100 mg of Compound 1. In some embodiments, thepharmaceutical composition comprises 125 mg of Compound 1. In someembodiments, the pharmaceutical composition comprises 150 mg ofCompound 1. In some embodiments, the pharmaceutical compositioncomprises 200 mg of Compound 1. In some embodiments, the pharmaceuticalcomposition comprises 250 mg of Compound 1. In some embodiments, thepharmaceutical composition comprises 300 mg of Compound 1. In someembodiments, the pharmaceutical composition comprises 400 mg of Compound1.

In one aspect, the invention provides a pharmaceutical compositioncomprising the following components:

(% w/w) Roller Compaction Granule Blend Compound 1 20-40Microcrystalline cellulose 30-50 Mannitol 10-30 Croscarmellose Sodium1-5 Sodium Lauryl Sulfate 0.1-2  Colloidal Silica 0.1-1  MagnesiumStearate 1-3 Tablet Composition Roller Compaction Granule Blend  99-99.9Magnesium Stearate 0.1-1 

In one aspect, the invention provides a pharmaceutical compositioncomprising the following components:

(% w/w) High Shear Granule Blend Compound 1 60-70 Microcrystallinecellulose  5-15 Croscarmellose Sodium 1-5 Sodium Lauryl Sulfate 0.1-2 Polyvinylpyrrolidone 1-5 Tablet Composition High Shear Granule Blend75-89 Microcrystalline cellulose 10-15 Croscarmellose Sodium 1-5Magnesium Stearate 0.1-5 

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) High Shear Granule Blend Compound 1 Form I 60-70Microcrystalline cellulose  5-15 Croscarmellose Sodium 1-5Polyvinylpyrrolidone 1-5 Sodium Lauryl Sulfate 0.1-2  Tablet CompositionHigh Shear Granule Blend 78-89 Microcrystalline cellulose 10-15Croscarmellose Sodium 1-5 Magnesium Stearate 0.1-2  Film Coated TabletCore Tablet Composition 95-99 Film Coat 1-5 Wax Trace

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) Roller Compaction Granule Blend Compound 1 Form I 30Microcrystalline cellulose 42.3 Mannitol 21.2 Croscarmellose Sodium 3Sodium Lauryl Sulfate 1 Colloidal Silica 0.5 Magnesium Stearate 2 TabletComposition Roller Compaction Granule Blend 99.5 Magnesium Stearate 0.5

In another aspect, the invention provides a pharmaceutical compositioncomprising the following components:

(% w/w) High Shear Granule Blend Compound 1 Form I 40-80Microcrystalline cellulose 20-40 Mannitol 10-15 Croscarmellose Sodium1-5 Polyvinylpyrrolidone  1-10 Sodium Lauryl Sulfate 0.1-2  TabletComposition High Shear Granule Blend 95-99 Croscarmellose Sodium 1-4Magnesium Stearate 0.1-1 

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) High Shear Granule Blend Compound 1 Form I 50 Microcrystallinecellulose 30 Mannitol 13 Croscarmellose Sodium 2 Polyvinylpyrrolidone 4Sodium Lauryl Sulfate 1 Tablet Composition High Shear Granule Blend 97.5Croscarmellose Sodium 2.0 Magnesium Stearate 0.5

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) High Shear Granule Blend Compound 1 Form I 60 Microcrystallinecellulose 20 Mannitol 13 Croscarmellose Sodium 2 Polyvinylpyrrolidone 4Sodium Lauryl Sulfate 1 Tablet Composition High Shear Granule Blend 97.5Croscarmellose Sodium 2.0 Magnesium Stearate 0.5

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) High Shear Granule Blend Compound 1 Form I 60 Microcrystallinecellulose 20 Mannitol 13 Croscarmellose Sodium 2 Polyvinylpyrrolidone 4Sodium Lauryl Sulfate 1 Tablet Composition High Shear Granule Blend 83Microcrystalline cellulose 14 Croscarmellose Sodium 2 Magnesium Stearate1

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) Twin Screw Granule Blend Compound 1 Form I 60 Microcrystallinecellulose 20 Mannitol 13 Croscarmellose Sodium 2 Polyvinylpyrrolidone 4Sodium Lauryl Sulfate 1 Tablet Composition Twin Screw Granule Blend 83Microcrystalline cellulose 14 Croscarmellose Sodium 2 Magnesium Stearate1

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) Twin Screw Wet Granule Blend Compound 1 Form I 80.0Microcrystalline cellulose 13.6 Croscarmellose Sodium 2.5Polyvinylpyrrolidone 3.1 Sodium Lauryl Sulfate 0.7 Tablet CompositionTwin Screw Granule Blend 83 Microcrystalline cellulose 12 CroscarmelloseSodium 4 Magnesium Stearate 1

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) Twin Screw Granule Blend Compound 1 Form I 80.0 Microcrystallinecellulose 13.6 Croscarmellose Sodium 2.5 Polyvinylpyrrolidone 3.1 SodiumLauryl Sulfate 0.7 Tablet Composition Twin Screw Granule Blend 83Microcrystalline cellulose 12 Croscarmellose Sodium 4 Magnesium Stearate1 Film Coated Tablet Core Tablet Composition 97 Film Coat 3 Wax Trace

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg High Shear Granule Blend Compound 1 Form I 200 Microcrystallinecellulose 66 Mannitol 43 Croscarmellose Sodium 7 Polyvinylpyrrolidone 13Sodium Lauryl Sulfate 3 Core Tablet Composition (200 mg dose) High ShearGranule Blend 332 Microcrystalline cellulose 56 Croscarmellose Sodium 8Magnesium Stearate 4 Film Coated Tablet (200 mg dose) Core TabletComposition 400 Film Coat 12 Wax trace

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg Twin Screw Granule Blend Compound 1 Form I 200 Microcrystallinecellulose 66 Mannitol 43 Croscarmellose Sodium 7 Polyvinylpyrrolidone 13Sodium Lauryl Sulfate 3 Core Tablet Composition (200 mg dose) Twin ScrewGranule Blend 332 Microcrystalline cellulose 56 Croscarmellose Sodium 8Magnesium Stearate 4

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg High Shear Granule Blend Compound 1 Form I 200 Microcrystallinecellulose 67 Mannitol 45 Croscarmellose Sodium 7 Polyvinylpyrrolidone10.4 Sodium Lauryl Sulfate 2.6 Core Tablet Composition (200 mg dose)High Shear Granule Blend 332 Microcrystalline cellulose 56Croscarmellose Sodium 8 Magnesium Stearate 4 Film Coated Tablet (200 mgdose) Core Tablet Composition 400 Film Coat 12 Wax trace

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg High Shear Granule Blend Compound 1 Form I 300 Microcrystallinecellulose 99 Mannitol 64.5 Croscarmellose Sodium 10.5Polyvinylpyrrolidone 19.5 Sodium Lauryl Sulfate 4.5 Core TabletComposition (300 mg dose) High Shear Granule Blend 498 Microcrystallinecellulose 84 Croscarmellose Sodium 12 Magnesium Stearate 6 Film CoatedTablet (300 mg dose) Core Tablet Composition 600 Film Coat 18 Wax trace

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg High Shear Granule Blend Compound 1 Form I 300 Microcrystallinecellulose 100.5 Mannitol 67.5 Croscarmellose Sodium 10.5Polyvinylpyrrolidone 15.6 Sodium Lauryl Sulfate 3.9 Core TabletComposition (300 mg dose) High Shear Granule Blend 498 Microcrystallinecellulose 84 Croscarmellose Sodium 12 Magnesium Stearate 6 Film CoatedTablet (300 mg dose) Core Tablet Composition 600 Film Coat 18 Wax trace

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) High Shear Granule Blend Compound 1 Form I 70 Microcrystallinecellulose 12 Mannitol 11 Croscarmellose Sodium 2 Polyvinylpyrrolidone 4Sodium Lauryl Sulfate 1 Tablet Composition High Shear Granule Blend 97.5Croscarmellose Sodium 2.0 Magnesium Stearate 0.5

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

(% w/w) High Shear Granule Blend Compound 1 Form I or Form II 61Microcrystalline cellulose 20.3 Mannitol 13.2 Croscarmellose Sodium 2Polyvinylpyrrolidone 2.7 Sodium Lauryl Sulfate 0.7 Tablet CompositionHigh Shear Granule Blend 83 Microcrystalline cellulose 14 CroscarmelloseSodium 2 Magnesium Stearate 1

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg High Shear Granule Blend Compound 1 Form I or Form II 100Microcrystalline cellulose 33.3 Mannitol 21.7 Croscarmellose Sodium 3.3Polyvinylpyrrolidone 4.4 Sodium Lauryl Sulfate 1.1 Core TabletComposition (100 mg dose) High Shear Granule Blend 163.9Microcrystalline cellulose 27.6 Croscarmellose Sodium 3.9 MagnesiumStearate 2.0

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg Twin Screw Granule Blend Compound 1 Form I 200 Microcrystallinecellulose 34.0 Croscarmellose Sodium 6.3 Polyvinylpyrrolidone 7.8 SodiumLauryl Sulfate 1.8 Core Tablet Composition (200 mg dose) Twin ScrewGranule Blend 249.9 Microcrystalline cellulose 36.1 CroscarmelloseSodium 12.0 Magnesium Stearate 3.0

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg Twin Screw Granule Blend Compound 1 Form I 400 Microcrystallinecellulose 68.0 Croscarmellose Sodium 12.6 Polyvinylpyrrolidone 15.6Sodium Lauryl Sulfate 3.6 Core Tablet Composition (400 mg dose) TwinScrew Granule Blend 499.8 Microcrystalline cellulose 72.2 CroscarmelloseSodium 24.0 Magnesium Stearate 6.0

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg Twin Screw Granule Blend Compound 1 Form I 200 Microcrystallinecellulose 34.0 Croscarmellose Sodium 6.3 Polyvinylpyrrolidone 7.8 SodiumLauryl Sulfate 1.8 Core Tablet Composition (200 mg dose) Twin ScrewGranule Blend 249.9 Microcrystalline cellulose 36.1 CroscarmelloseSodium 12.0 Magnesium Stearate 3.0 Film Coated Tablet (200 mg dose, 310mg total) Core Tablet Composition 301 Film Coat 9.0 Wax trace

In another embodiment, the invention provides a pharmaceuticalcomposition comprising the following components:

mg Twin Screw Granule Blend Compound 1 Form I 400 Microcrystallinecellulose 68.0 Croscarmellose Sodium 12.6 Polyvinylpyrrolidone 15.6Sodium Lauryl Sulfate 3.6 Core Tablet Composition (400 mg dose) TwinScrew Granule Blend 499.8 Microcrystalline cellulose 72.2 CroscarmelloseSodium 24.0 Magnesium Stearate 6.0 Film Coated Tablet (400 mg dose, 620mg total) Core Tablet Composition 602 Film Coat 18.0 Wax trace

In another aspect, the invention provides a pharmaceutical compositionin the form of a tablet that comprises Compound 1, and one or morepharmaceutically acceptable excipients, for example, a filler, adisintegrant, a surfactant, a diluent, a binder, a glidant, and alubricant and any combination thereof, where the tablet has adissolution of at least about 50% in about 30 minutes. In anotherembodiment, the dissolution rate is at least about 75% in about 30minutes. In another embodiment, the dissolution rate is at least about90% in about 30 minutes.

In another aspect, the invention provides a pharmaceutical compositionconsisting of a tablet that comprises a powder blend or granulescomprising Compound 1; and, one or more pharmaceutically acceptableexcipients, for example, a filler, a disintegrant, a surfactant, adiluent, a binder, a glidant, and a lubricant, wherein the tablet has ahardness of at least about 5 kP (kP=kilo Ponds; 1 kP=˜9.8 N). In anotherembodiment, the tablet has a target friability of less than 1.0% after400 revolutions. In another aspect, the invention provides apharmaceutical composition consisting of a tablet that comprises apowder blend or granules comprising Compound 1 Form II, Compound 1; and,one or more pharmaceutically acceptable excipients, for example, afiller, a disintegrant, a surfactant, a diluent, a binder, a glidant,and a lubricant, wherein the tablet has a hardness of at least about 5kP (kP=kilo Ponds; 1 kP=˜9.8 N). In another embodiment, the tablet has atarget friability of less than 1.0% after 400 revolutions.

In another aspect, the invention provides a pharmaceutical compositionas described herein further comprising an additional therapeutic agent.In some embodiments, the additional therapeutic agent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3 -carboxamide.

In another aspect, the invention provides a method of treating a CFTRmediated disease in a mammal comprising administering to the mammal aneffective amount of a pharmaceutical composition as described herein. Insome embodiments, the CFTR mediated disease is cystic fibrosis,emphysema, COPD, or osteoporosis. In other embodiments, the CFTRmediated disease is cystic fibrosis. This method may further compriseadministering an additional therapeutic agent, wherein in someembodiments, the additional therapeutic agent is selected from amucolytic agent, bronchodilator, an anti-biotic, an anti-infectiveagent, an anti-inflammatory agent, a CFTR potentiator, or a nutritionalagent. In another embodiment, the additional therapeutic agent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.In another embodiment, the patient has a F508del-CFTR mutation. Inanother embodiment, the patient is homozygous for F508del. In anotherembodiment, the patient is heterozygous for F508del.

In another aspect, the invention features a kit comprising a tablet ofthe present invention, and a separate therapeutic agent orpharmaceutical composition thereof. In another embodiment, the Compound1 in the tablet is in Form I. In another embodiment, the therapeuticagent is a cystic fibrosis corrector other than Compound 1. In anotherembodiment, the therapeutic agent is a cystic fibrosis potentiator. Inanother embodiment, the therapeutic agent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.In another embodiment, the tablet and the therapeutic agent are inseparate containers. In another embodiment, the separate containers arebottles. In another embodiment, the separate containers are vials. Inanother embodiment, the separate containers are blister packs.

In another aspect, the invention provides a process for making thepharmaceutical compositions described herein by a roller compactionprocess comprising the steps of screening and weighing Compound 1 andexcipients; blending Compound 1 and excipients for a suitable amount oftime; roller compacting the blend into ribbons and milling the ribbonsinto granules; blending the granules with extra-granular excipients fora suitable amount of time; compressing the blend into tablets; coatingthe tablets; and, optionally, printing a monogram on one or both tabletfaces.

In another aspect, the invention provides a process for making thepharmaceutical compositions described herein by a high shear granulationprocess comprising the steps of screening and weighing Compound 1 andexcipients; mixing Compound 1 and excipients while adding a granulationfluid comprising surfactant and a binder at a suitable mixing speed fora suitable amount of time and chopping the mixture into granules; dryingthe granules; blending the granules with extra-granular excipients for asuitable amount of time; compressing the blend into tablets; coating thetablets; and, optionally, printing a monogram on one or both tabletfaces.

In another aspect, the invention provides a continuous orsemi-continuous process for making the pharmaceutical compositionsdescribed herein by a twin screw wet granulation process comprising thesteps of screening and weighing Compound 1 and excipients; mixingCompound 1 and excipients in a blender and feeding the blend into acontinuous granulator while adding a granulation fluid comprisingsurfactant and a binder at a suitable rate for a suitable amount of timeand chopping the mixture into granules; drying the granules; blendingthe granules with extra-granular excipients for a suitable amount oftime; compressing the blend into tablets; coating the tablets; and,optionally, printing a monogram on one or both tablet faces.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an X-ray diffraction pattern calculated from a single crystalstructure of Compound 1 Form I.

FIG. 2 is an actual X-ray powder diffraction pattern of Compound 1 FormI.

FIG. 3 is an X-ray powder diffraction pattern of Compound 1 Form II.

FIG. 4 provides X-ray diffraction patterns of Compound 1 Form II'sselected from:

1) Compound 1 Form II, Methanol Solvate;

2) Compound 1 Form II, Ethanol Solvate;

3) Compound 1 Form II, Acetone Solvate;

4) Compound 1 Form II, 2-Propanol Solvate;

5) Compound 1 Form II, Acetonitrile Solvate;

6) Compound 1 Form II, Tetrahydrofuran Solvate;

7) Compound 1 Form II, Methyl Acetate Solvate;

8) Compound 1 Form II, 2-Butanone Solvate;

9) Compound 1 Form II, Ethyl Formate Solvate; and

10) Compound 1 Form II, 2-Methyltetrahydrofuran Solvate.

FIG. 5 provides an X-ray diffraction pattern of Compound 1 Form II,Methanol Solvate.

FIG. 6 provides an X-ray diffraction pattern of Compound 1 Form II,Ethanol Solvate.

FIG. 7 provides an X-ray diffraction pattern of Compound 1 Form II,Acetone Solvate.

FIG. 8 provides an X-ray diffraction pattern of Compound 1 Form II,2-Propanol Solvate.

FIG. 9 provides an X-ray diffraction pattern of Compound 1 Form II,Acetonitrile Solvate.

FIG. 10 provides an X-ray diffraction pattern of Compound 1 Form II,Tetrahydrofuran Solvate.

FIG. 11 provides an X-ray diffraction pattern of Compound 1 Form II,Methyl Acetate Solvate.

FIG. 12 provides an X-ray diffraction pattern of Compound 1 Form II,2-Butanone Solvate.

FIG. 13 provides an X-ray diffraction pattern of Compound 1 Form II,Ethyl Formate Solvate.

FIG. 14 provides an X-ray diffraction pattern of Compound 1 Form II,2-Methyltetrahydrofuran Solvate.

FIG. 15 is a differential scanning calorimetry (DSC) trace of Compound 1Form II, Acetone Solvate.

FIG. 16 is a Thermogravimetric analysis (TGA) plot of Compound 1 FormII, Acetone Solvate.

FIG. 17 is a conformational image of Compound 1 Form II, Acetone Solvatebased on single crystal X-ray analysis.

FIG. 18 is a conformational image of the dimer of Compound 1 HCl SaltForm A.

FIG. 19 is an X-ray diffraction pattern of Compound 1 HCl Salt Form Acalculated from the crystal structure.

FIG. 20 is an ¹HNMR spectrum of Compound 1.

FIG. 21 is an ¹HNMR spectrum of Compound 1 HCl salt.

FIG. 22 is a differential scanning calorimetry (DSC) trace of Compound 1Form I.

FIG. 23 is a conformational picture of Compound 1 Form I based on singlecrystal X-ray analysis.

FIG. 24 is a conformational image of Compound 1 Form II, AcetoneSolvate, based on single crystal X-ray analysis.

FIG. 25 is a solid state ¹³C NMR spectrum (15.0 kHz spinning) ofCompound 1 Form II, Acetone Solvate.

FIG. 26 is a solid state ¹⁹F NMR spectrum (12.5 kHz spinning) ofCompound 1 Form II, Acetone Solvate.

FIG. 27 is an X-ray diffraction pattern of Compound 1 HCl Salt Form Acalculated from the crystal structure.

FIG. 28 is a graph depicting Compound 1 pH gradient dissolution profilesfor a tablet made by a high shear granulation (HSG) process and a twinscrew wet granulation (TSWG) process (LOD stands for loss on drying, ameasure to define the amount of water in a powder/granule).

DETAILED DESCRIPTION Definitions

As used herein, “CFTR” stands for cystic fibrosis transmembraneconductance regulator.

As used herein, a “ΔF508” or “F508def” is a specific mutation within theCFTR protein. The mutation is a deletion of the three nucleotides thatcomprise the codon for amino acid phenylalanine at position 508,resulting in CFTR protein that lacks this particular phenylalanine.

As used herein, a patient who is “homozygous” for a particular mutation,e.g. F508del, has the same mutation on both alleles.

As used herein, a patient who is “heterozygous” for a particularmutation, e.g. F508del, has this mutation on one allele, and a differentmutation on the other allele.

As used herein, the term “CFTR corrector” refers to a compound thataugments or induces the amount of functional CFTR protein to the cellsurface, resulting in increased functional activity.

As used herein, the term “CFTR potentiator” refers to a compound thataugments or induces the channel activity of CFTR protein located at thecell surface, resulting in increased functional activity.

As used herein, the term “active pharmaceutical ingredient” or “API”refers to a biologically active compound. Exemplary APIs include3-(6-(1(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Compound 1).

The terms “solid form”, “solid forms” and related terms, when usedherein to refer to 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl) benzoic acid (Compound1), refer to a solid form e.g. crystals and the like, comprisingCompound 1 which is not predominantly in a liquid or a gaseous state.

As used herein, the term “substantially amorphous” refers to a solidmaterial having little or no long range order in the position of itsmolecules. For example, substantially amorphous materials have less thanabout 15% crystallinity (e.g., less than about 10% crystallinity or lessthan about 5% crystallinity). It is also noted that the term‘substantially amorphous’ includes the descriptor, ‘amorphous’, whichrefers to materials having no (0%) crystallinity.

As used herein, the term “substantially crystalline” (as in the phrasesubstantially crystalline Compound 1 Form I, Compound 1 Form II, orCompound 1 HCl Salt Form A) refers to a solid material havingpredominantly long range order in the position of its molecules. Forexample, substantially crystalline materials have more than about 85%crystallinity (e.g., more than about 90% crystallinity or more thanabout 95% crystallinity). It is also noted that the term ‘substantiallycrystalline’ includes the descriptor, ‘crystalline’, which refers tomaterials having 100% crystallinity.

The term “crystalline” and related terms used herein, when used todescribe a substance, component, product, or form, means that thesubstance, component or product is substantially crystalline asdetermined by X-ray diffraction. (See, e.g., Remington: The Science andPractice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins,Baltimore, Md. (2003); The United States Pharmacopeia, 23^(rd) ed.,1843-1844 (1995)).

As used herein, the term “composition” generally refers to a compositionof two or more components, usually one or more drugs (e.g., one drug(e.g., Compound 1 Form I, Compound 1 Form II, or Compound 1 HCl SaltForm A)) and one or more pharmaceutical excipients.

As used herein, the term “solid dosage form” generally refers to apharmaceutical composition, which when used in an oral mode ofadministration include capsules, tablets, pills, powders and granules.In such solid dosage forms, the active compound is mixed with at leastone inert, pharmaceutically acceptable excipient or carrier.

As used herein, an “excipient” includes functional and non-functionalingredients in a pharmaceutical composition.

As used herein, a “disintegrant” is an excipient that hydrates apharmaceutical composition and aids in tablet dispersion. As usedherein, a “diluent” or “filler” is an excipient that adds bulkiness to apharmaceutical composition.

As used herein, a “surfactant” is an excipient that impartspharmaceutical compositions with enhanced solubility and/or wetability.

As used herein, a “binder” is an excipient that imparts a pharmaceuticalcomposition with enhanced cohesion or tensile strength (e.g., hardness).

As used herein, a “glidant” is an excipient that imparts apharmaceutical compositions with enhanced flow properties.

As used herein, a “colorant” is an excipient that imparts apharmaceutical composition with a desired color. Examples of colorantsinclude commercially available pigments such as FD&C Blue #1 AluminumLake, FD&C Blue #2, other FD&C Blue colors, titanium dioxide, ironoxide, and/or combinations thereof In one embodiment, the pharmaceuticalcomposition provided by the invention is purple.

As used herein, a “lubricant” is an excipient that is added topharmaceutical compositions that are pressed into tablets. The lubricantaids in compaction of granules into tablets and ejection of a tablet ofa pharmaceutical composition from a die press.

As used herein, “cubic centimeter” and “cc” are used interchangeably torepresent a unit of volume. Note that 1 cc=1 mL.

As used herein, “kiloPond” and “kP” are used interchangeably and referto the measure of force where a kP=approximately 9.8 Newtons.

As used herein, “friability” refers to the property of a tablet toremain intact and withhold its form despite an external force ofpressure. Friability can be quantified using the mathematical expressionpresented in equation 1:

$\begin{matrix}{{\%\mspace{14mu}{friability}} = {100 \times \frac{\left( {W_{0} - W_{f}} \right)}{W_{0}}}} & (1)\end{matrix}$

wherein W₀ is the original weight of the tablet and W_(f) is the finalweight of the tablet after it is put through the friabilator. Friabilityis measured using a standard USP testing apparatus that tumblesexperimental tablets for 100 or 400 revolutions. Some tablets of theinvention have a friability of less than 5.0%. In another embodiment,the friability is less than 2.0%. In another embodiment, the targetfriability is less than 1.0% after 400 revolutions.

As used herein, “mean particle diameter” is the average particlediameter as measured using techniques such as laser light scattering,image analysis, or sieve analysis. In one embodiment, the granules usedto prepare the pharmaceutical compositions provided by the inventionhave a mean particle diameter of less than 1.0 mm.

As used herein, “bulk density” is the mass of particles of materialdivided by the total volume the particles occupy. The total volumeincludes particle volume, inter-particle void volume and internal porevolume. Bulk density is not an intrinsic property of a material; it canchange depending on how the material is processed. In one embodiment,the granules used to prepare the pharmaceutical compositions provided bythe invention have a bulk density of about 0.5-0.7 g/cc.

An effective amount or “therapeutically effective amount” of a drugcompound of the invention may vary according to factors such as thedisease state, age, and weight of the subject, and the ability of thecompound of the invention to elicit a desired response in the subject.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (e.g., side effects) of the compound of theinvention are outweighed by the therapeutically beneficial effects.

As used herein, and unless otherwise specified, the terms“therapeutically effective amount” and “effective amount” of a compoundmean an amount sufficient to provide a therapeutic benefit in thetreatment or management of a disease or disorder, or to delay orminimize one or more symptoms associated with the disease or disorder. A“therapeutically effective amount” and “effective amount” of a compoundmean an amount of therapeutic agent, alone or in combination with one ormore other agent(s), which provides a therapeutic benefit in thetreatment or management of the disease or disorder. The terms“therapeutically effective amount” and “effective amount” can encompassan amount that improves overall therapy, reduces or avoids symptoms orcauses of disease or disorder, or enhances the therapeutic efficacy ofanother therapeutic agent.

“Substantially pure” as used in the phrase “substantially pure Compound1 Form I, Compound 1 Form II, or Compound 1 HCl Salt Form A,” meansgreater than about 90% purity. In another embodiment, substantially purerefers to greater than about 95% purity. In another embodiment,substantially pure refers to greater than about 98% purity. In anotherembodiment, substantially pure refers to greater than about 99% purity.

With respect to Compound 1 (e.g., Compound 1 Form I, Compound 1 Form II,Compound 1 HCl Salt Form A), the terms “about” and “approximately”, whenused in connection with doses, amounts, or weight percent of ingredientsof a composition or a dosage form, mean a dose, amount, or weightpercent that is recognized by one of ordinary skill in the art toprovide a pharmacological effect equivalent to that obtained from thespecified dose, amount, or weight percent. Specifically the term “about”or “approximately” means an acceptable error for a particular value asdetermined by one of ordinary skill in the art, which depends in part onhow the value is measured or determined. In certain embodiments, theterm “about” or “approximately” means within 1, 2, 3, or 4 standarddeviations. In certain embodiments, the term “about” or “approximately”means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,1%, 0.5%, 0.1%, or 0.05% of a given value or range.

Unless otherwise specified, the term “Compound 1” includes, but is notlimited to, the solid forms of Compound 1 as described herein, e.g.Compound 1 Form I, Compound 1 Form II, or Compound 1 HCl Salt Form A, aswell as combinations thereof.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions, pharmaceuticalformulations and solid dosage forms comprising Compound 1 which may bein substantially crystalline form. In some embodiments, Compound 1 is incrystalline Form I (Compound 1 Form I). In some embodiments, Compound 1is in crystalline Form II (Compound 1 Form II). In some embodiments,Compound 1 is in crystalline HCl salt form (Compound 1 HCl Salt Form A).In some embodiments of this aspect, the amount of Compound 1 that ispresent in the pharmaceutical composition is 25 mg, 50 mg, 75 mg, 100mg, 125 mg, 150 mg, 200 mg, 250 mg, or 400 mg. In some embodiments ofthis aspect, weight/weight relative percent of Compound 1 that ispresent in the pharmaceutical composition is from 10 to 75 percent. Inthese and other embodiments,3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid is present assubstantially pure Compound 1. “Substantially pure” means greater thanninety percent pure; preferably greater than 95 percent pure; morepreferably greater than 99.5 percent pure (i.e., not mixed with othercrystalline forms of Compound 1).

Thus in one aspect, the invention provides a pharmaceutical compositioncomprising:

a. Compound 1;

b. a filler;

c. a disintegrant;

d. a surfactant;

e. a diluent;

f. a lubricant; and

g. a glidant or a binder.

In one embodiment of this aspect, the pharmaceutical compositioncomprises 25 mg of Compound 1. In another embodiment of this aspect, thepharmaceutical composition comprises 50 mg of Compound 1. In anotherembodiment of this aspect, the pharmaceutical composition comprises 100mg of Compound 1. In another embodiment of this aspect, thepharmaceutical composition comprises 125 mg of Compound 1. In anotherembodiment of this aspect, the pharmaceutical composition comprises 150mg of Compound 1. In another embodiment of this aspect, thepharmaceutical composition comprises 200 mg of Compound 1. In anotherembodiment of this aspect, the pharmaceutical composition comprises 250mg of Compound 1. In another embodiment of this aspect, thepharmaceutical composition comprises 300 mg of Compound 1. In anotherembodiment of this aspect, the pharmaceutical composition comprises 400mg of Compound 1.

In some embodiments, the pharmaceutical compositions comprises Compound1, wherein Compound 1 is present in an amount of at least 15 wt % (e.g.,at least 20 wt %, at least 30 wt %, at least 40 wt %, at least 50 wt %,at least 60 wt %, or at least 70 wt %) by weight of the composition.

In some embodiments, the pharmaceutical composition comprises Compound1, a filler, a diluent, a disintegrant, a surfactant, a glidant, and alubricant. In this embodiment, the composition comprises from about 20wt % to about 50 wt % (e.g., about 25-35 wt %) of Compound 1 by weightof the composition, and more typically, from 25 wt % to about 45 wt %(e.g., about 28-32 wt %) of Compound 1 by weight of the composition.

In some embodiments, the pharmaceutical composition comprises Compound1, a filler, a diluent, a disintegrant, a surfactant, a binder, and alubricant. In this embodiment, the composition comprises from about 30wt % to about 60 wt % (e.g., about 40-55 wt %) of Compound 1 by weightof the composition, and more typically from 35 wt % to about 70 wt %(e.g., about 45-55 wt %) of Compound 1 by weight of the composition.

The concentration of Compound 1 in the composition depends on severalfactors such as the amount of pharmaceutical composition needed toprovide a desired amount of Compound 1 and the desired dissolutionprofile of the pharmaceutical composition.

In another embodiment, the pharmaceutical composition comprises Compound1, in which Compound 1 in its solid form has a mean particle diameter,measured by light scattering (e.g., using a Malvern Mastersizeravailable from Malvern Instruments in England) of 0.1 microns to 10microns. In another embodiment, the particle size of Compound 1 is 1micron to 5 microns. In another embodiment, Compound 1 has a particlesize D50 of 2.0 microns.

As indicated, in addition to Compound 1, in some embodiments of theinvention, the pharmaceutical compositions which are oral formulationsalso comprise one or more excipients such as fillers, disintegrants,surfactants, diluents, binders, glidants, lubricants, colorants, orfragrances and any combination thereof.

Fillers suitable for the invention are compatible with the ingredientsof the pharmaceutical composition, i.e., they do not substantiallyreduce the solubility, the hardness, the chemical stability, thephysical stability, or the biological activity of the pharmaceuticalcomposition. Exemplary fillers include: celluloses, modified celluloses,(e.g. sodium carboxymethyl cellulose, ethyl cellulose hydroxymethylcellulose, hydroxypropylcellulose), cellulose acetate, microcrystallinecellulose, calcium phosphates, dibasic calcium phosphate, starches (e.g.corn starch, potato starch), sugars (e.g., sorbitol) lactose, sucrose,or the like), or any combination thereof.

Thus, in one embodiment, the pharmaceutical composition comprises atleast one filler in an amount of at least 5 wt % (e.g., at least about20 wt %, at least about 30 wt %, or at least about 40 wt %) by weight ofthe composition. For example, the pharmaceutical composition comprisesfrom about 10 wt % to about 60 wt % (e.g., from about 20 wt % to about55 wt %, from about 25 wt % to about 50 wt %, or from about 27 wt % toabout 45 wt %) of filler, by weight of the composition. In anotherexample, the pharmaceutical composition comprises at least about 20 wt %(e.g., at least 30 wt % or at least 40 wt %) of microcrystallinecellulose, for example MCC Avicel PH102, by weight of the composition.In yet another example, the pharmaceutical composition comprises fromabout 10 wt % to about 60 wt % (e.g., from about 20 wt % to about 55 wt% or from about 25 wt % to about 45 wt %) of microcellulose, by weightof the composition.

Disintegrants suitable for the invention enhance the dispersal of thepharmaceutical composition and are compatible with the ingredients ofthe pharmaceutical composition, i.e., they do not substantially reducethe chemical stability, the physical stability, the hardness, or thebiological activity of the pharmaceutical composition. Exemplarydisintegrants include croscarmellose sodium, sodium starch glycolate, ora combination thereof

Thus, in one embodiment, the pharmaceutical composition comprisesdisintegrant in an amount of about 10 wt % or less (e.g., about 7 wt %or less, about 6 wt % or less, or about 5 wt % or less) by weight of thecomposition. For example, the pharmaceutical composition comprises fromabout 1 wt % to about 10 wt % (e.g., from about 1.5 wt % to about 7.5 wt% or from about 2.5 wt % to about 6 wt %) of disintegrant, by weight ofthe composition. In another example, the pharmaceutical compositioncomprises about 10 wt % or less (e.g., 7 wt % or less, 6 wt % or less,or 5 wt % or less) of croscarmellose sodium, by weight of thecomposition. In yet another example, the pharmaceutical compositioncomprises from about 1 wt % to about 10 wt % (e.g., from about 1.5 wt %to about 7.5 wt % or from about 2.5 wt % to about 6 wt %) ofcroscarmellose sodium, by weight of the composition. In some examples,the pharmaceutical composition comprises from about 0.1% to about 10 wt% (e.g., from about 0.5 wt % to about 7.5 wt % or from about 1.5 wt % toabout 6 wt %) of disintegrant, by weight of the composition. In stillother examples, the pharmaceutical composition comprises from about 0.5%to about 10 wt % (e.g., from about 1.5 wt % to about 7.5 wt % or fromabout 2.5 wt % to about 6 wt %) of disintegrant, by weight of thecomposition.

Surfactants suitable for the invention enhance the wettability of thepharmaceutical composition and are compatible with the ingredients ofthe pharmaceutical composition, i.e., they do not substantially reducethe chemical stability, the physical stability, the hardness, or thebiological activity of the pharmaceutical composition. Exemplarysurfactants include sodium lauryl sulfate (SLS), sodium stearyl fumarate(SSF), polyoxyethylene 20 sorbitan mono-oleate (e.g., Tween™), anycombination thereof, or the like.

Thus, in one embodiment, the pharmaceutical composition comprises asurfactant in an amount of about 10 wt % or less (e.g., about 5 wt % orless, about 2 wt % or less, about 1 wt % or less, about 0.8 wt % orless, or about 0.6 wt % or less) by weight of the composition. Forexample, the pharmaceutical composition includes from about 10 wt % toabout 0.1 wt % (e.g., from about 5 wt % to about 0.2 wt % or from about2 wt % to about 0.3 wt %) of surfactant, by weight of the composition.In another example, the pharmaceutical composition comprises 10 wt % orless (e.g., about 5 wt % or less, about 2 wt % or less, about 1 wt % orless, about 0.8 wt % or less, or about 0.6 wt % or less) of sodiumlauryl sulfate, by weight of the composition. In yet another example,the pharmaceutical composition comprises from about 10 wt % to about 0.1wt % (e.g., from about 5 wt % to about 0.2 wt % or from about 2 wt % toabout 0.3 wt %) of sodium lauryl sulfate, by weight of the composition.

Binders suitable for the invention enhance the tablet strength of thepharmaceutical composition and are compatible with the ingredients ofthe pharmaceutical composition, i.e., they do not substantially reducethe chemical stability, the physical stability, or the biologicalactivity of the pharmaceutical composition. Exemplary binders includepolyvinylpyrrolidone, dibasic calcium phosphate, sucrose, corn (maize)starch, modified cellulose (e.g., hydroxymethyl cellulose), or anycombination thereof

Thus, in one embodiment, the pharmaceutical composition comprises abinder in an amount of at least about 0.1 wt % (e.g., at least about 1wt %, at least about 3 wt %, at least about 4 wt %, or at least about 5wt %) by weight of the composition. For example, the pharmaceuticalcomposition comprises from about 0.1 wt % to about 10 wt % (e.g., fromabout 1 wt % to about 10 wt % or from about 2 wt % to about 7 wt %) ofbinder, by weight of the composition. In another example, thepharmaceutical composition comprises at least about 0.1 wt % (e.g., atleast about 1 wt %, at least about 2 wt %, at least about 3 wt %, or atleast about 4 wt %) of polyvinylpyrrolidone, by weight of thecomposition. In yet another example, the pharmaceutical compositioncomprises a glidant in an amount ranging from about 0.1 wt % to about 10wt % (e.g., from about 1 wt % to about 8 wt % or from about 2 wt % toabout 5 wt %) of polyvinylpyrrolidone, by weight of the composition.

Diluents suitable for the invention may add necessary bulk to aformulation to prepare tablets of the desired size and are generallycompatible with the ingredients of the pharmaceutical composition, i.e.,they do not substantially reduce the solubility, the hardness, thechemical stability, the physical stability, or the biological activityof the pharmaceutical composition. Exemplary diluents include: sugars,for example, confectioner's sugar, compressible sugar, dextrates,dextrin, dextrose, lactose, mannitol, sorbitol, cellulose, and modifiedcelluloses, for example, powdered cellulose, talc, calcium phosphate,starch, or any combination thereof.

Thus, in one embodiment, the pharmaceutical composition comprises adiluent in an amount of 40 wt % or less (e.g., 35 wt % or less, 30 wt %or less, or 25 wt % or less, or 20 wt % or less, or 15 wt % or less, or10 wt % or less) by weight of the composition. For example, thepharmaceutical composition comprises from about 40 wt % to about 1 wt %(e.g., from about 35 wt % to about 5 wt % or from about 30 wt % to about7 wt %, from about 25 wt % to about 10 wt %, from about 20 wt % to about15 wt %) of diluent, by weight of the composition. In another example,the pharmaceutical composition comprises 40 wt % or less (e.g., 35 wt %or less, 25 wt % or less, or 15 wt % or less) of mannitol, by weight ofthe composition. In yet another example, the pharmaceutical compositioncomprises from about 35 wt % to about 1 wt % (e.g., from about 30 wt %to about 5 wt % or from about 25 wt % to about 10 wt %) of mannitol, byweight of the composition.

Glidants suitable for the invention enhance the flow properties of thepharmaceutical composition and are compatible with the ingredients ofthe pharmaceutical composition, i.e., they do not substantially reducethe solubility, the hardness, the chemical stability, the physicalstability, or the biological activity of the pharmaceutical composition.Exemplary glidants include colloidal silicon dioxide, talc, or acombination thereof.

Thus, in one embodiment, the pharmaceutical composition comprises aglidant in an amount of 2 wt % or less (e.g., 1.75 wt %, 1.25 wt % orless, or 1.00 wt % or less) by weight of the composition. For example,the pharmaceutical composition comprises from about 2 wt % to about 0.05wt % (e.g., from about 1.5 wt % to about 0.07 wt % or from about 1.0 wt% to about 0.09 wt %) of glidant, by weight of the composition. Inanother example, the pharmaceutical composition comprises 2 wt % or less(e.g., 1.75 wt %, 1.25 wt % or less, or 1.00 wt % or less) of colloidalsilicon dioxide, by weight of the composition. In yet another example,the pharmaceutical composition comprises from about 2 wt % to about 0.05wt % (e.g., from about 1.5 wt % to about 0.07 wt % or from about 1.0 wt% to about 0.09 wt %) of colloidal silicon dioxide, by weight of thecomposition.

In some embodiments, the pharmaceutical composition can include an oralsolid pharmaceutical dosage form which can comprise a lubricant that canprevent adhesion of a granulate-bead admixture to a surface (e.g., asurface of a mixing bowl, a compression die and/or punch). A lubricantcan also reduce interparticle friction within the granulate and improvethe compression and ejection of compressed pharmaceutical compositionsfrom a die press. The lubricant is also compatible with the ingredientsof the pharmaceutical composition, i.e., they do not substantiallyreduce the solubility, the hardness, or the biological activity of thepharmaceutical composition. Exemplary lubricants include magnesiumstearate, calcium stearate, zinc stearate, sodium stearate, stearicacid, aluminum stearate, leucine, glyceryl behenate, hydrogenatedvegetable oil or any combination thereof. In one embodiment, thepharmaceutical composition comprises a lubricant in an amount of 5 wt %or less (e.g., 4.75 wt %, 4.0 wt % or less, or 3.00 wt % or less, or 2.0wt % or less) by weight of the composition. For example, thepharmaceutical composition comprises from about 5 wt % to about 0.10 wt% (e.g., from about 4.5 wt % to about 0.5 wt % or from about 3 wt % toabout 1 wt %) of lubricant, by weight of the composition. In anotherexample, the pharmaceutical composition comprises 5 wt % or less (e.g.,4.0 wt % or less, 3.0 wt % or less, or 2.0 wt % or less, or 1.0 wt % orless) of magnesium stearate, by weight of the composition. In yetanother example, the pharmaceutical composition comprises from about 5wt % to about 0.10 wt % (e.g., from about 4.5 wt % to about 0.15 wt % orfrom about 3.0 wt % to about 0.50 wt %) of magnesium stearate, by weightof the composition.

Pharmaceutical compositions of the invention can optionally comprise oneor more colorants, flavors, and/or fragrances to enhance the visualappeal, taste, and/or scent of the composition. Suitable colorants,flavors, or fragrances are compatible with the ingredients of thepharmaceutical composition, i.e., they do not substantially reduce thesolubility, the chemical stability, the physical stability, thehardness, or the biological activity of the pharmaceutical composition.In one embodiment, the pharmaceutical composition comprises a colorant,a flavor, and/or a fragrance. In one embodiment, the pharmaceuticalcompositions provided by the invention are purple.

In some embodiments, the pharmaceutical composition includes or can bemade into tablets and the tablets can be coated with a colorant andoptionally labeled with a logo, other image and/or text using a suitableink. In still other embodiments, the pharmaceutical composition includesor can be made into tablets and the tablets can be coated with acolorant, waxed, and optionally labeled with a logo, other image and/ortext using a suitable ink. Suitable colorants and inks are compatiblewith the ingredients of the pharmaceutical composition, i.e., they donot substantially reduce the solubility, the chemical stability, thephysical stability, the hardness, or the biological activity of thepharmaceutical composition. The suitable colorants and inks can be anycolor and are water based or solvent based. In one embodiment, tabletsmade from the pharmaceutical composition are coated with a colorant andthen labeled with a logo, other image, and/or text using a suitable ink.For example, tablets comprising pharmaceutical composition as describedherein can be coated with about 3 wt % (e.g., less than about 6 wt % orless than about 4 wt %) of film coating comprising a colorant. Thecolored tablets can be labeled with a logo and text indicating thestrength of the active ingredient in the tablet using a suitable ink. Inanother example, tablets comprising pharmaceutical composition asdescribed herein can be coated with about 3 wt % (e.g., less than about6 wt % or less than about 4 wt %) of a film coating comprising acolorant.

In another embodiment, tablets made from the pharmaceutical compositionare coated with a colorant, waxed, and then labeled with a logo, otherimage, and/or text using a suitable ink. For example, tablets comprisingpharmaceutical composition as described herein can be coated with about3 wt % (e.g., less than about 6 wt % or less than about 4 wt %) of filmcoating comprising a colorant. The colored tablets can be waxed withCarnauba wax powder weighed out in the amount of about 0.01% w/w of thestarting tablet core weight. The waxed tablets can be labeled with alogo and text indicating the strength of the active ingredient in thetablet using a suitable ink. In another example, tablets comprisingpharmaceutical composition as described herein can be coated with about3 wt % (e.g., less than about 6 wt % or less than about 4 wt %) of afilm coating comprising a colorant The colored tablets can be waxed withCarnauba wax powder weighed out in the amount of about 0.01% w/w of thestarting tablet core weight. The waxed tablets can be labeled with alogo and text indicating the strength of the active ingredient in thetablet using a pharmaceutical grade ink such as a black ink (e.g.,Opacode® S-1-17823, a solvent based ink, commercially available fromColorcon, Inc. of West Point, Pa.).

One exemplary pharmaceutical composition comprises from about 15 wt % toabout 70 wt % (e.g., from about 15 wt % to about 60 wt %, from about 15wt % to about 50 wt %, or from about 15 wt % to about 40 wt %, or fromabout 20 wt % to about 70 wt %, or from about 30 wt % to about 70 wt %,or from about 40 wt % to about 70 wt %, or from about 50 wt % to about70 wt %) of Compound 1, by weight of the composition. The aforementionedcompositions can also include one or more pharmaceutically acceptableexcipients, for example, from about 20 wt % to about 50 wt % of afiller; from about 1 wt % to about 5 wt % of a disintegrant; from about2 wt % to about 0.3 wt % of a surfactant; from about 0.1 wt % to about 5wt % of a binder; from about 1 wt % to about 30 wt % of a diluent; fromabout 2 wt % to about 0.05 wt % of a glidant; and from about 5 wt % toabout 0.1 wt % of a lubricant. Or, the pharmaceutical compositioncomprises a composition containing from about 15 wt % to about 70 wt %(e.g., from about 20 wt % to about 40 wt %, from about 25 wt % to about60 wt %, or from about 30 wt % to about 55 wt %) of Compound 1, byweight of the composition; and one or more excipients, for example, fromabout 20 wt % to about 50 wt % of a filler; from about 1 wt % to about 5wt % of a disintegrant; from about 2 wt % to about 0.3 wt % of asurfactant; from about 0.1 wt % to about 5 wt % of a binder; from about1 wt % to about 30 wt % of a diluent; from about 2 wt % to about 0.05 wt% of a glidant; and from about 5 wt % to about 0.1 wt % of a lubricant.

Another exemplary pharmaceutical composition comprises from about 15 wt% to about 70 wt % (e.g., from about 15 wt % to about 60 wt %, fromabout 15 wt % to about 50 wt %, or from about 15 wt % to about 40 wt %or from about 20 wt % to about 70 wt %, or from about 30 wt % to about70 wt %, or from about 40 wt % to about 70 wt %, or from about 50 wt %to about 70 wt %) of Compound 1 by weight of the composition, and one ormore excipients, for example, from about 20 wt % to about 50 wt % of afiller; from about 1 wt % to about 5 wt % of a disintegrant; from about2 wt % to about 0.3 wt % of a surfactant; from about 0.1 wt % to about 5wt % of a binder; from about 1 wt % to about 30 wt % of a diluent; fromabout 2 wt % to about 0.05 wt % of a glidant; and from about 2 wt % toabout 0.1 wt % of a lubricant.

Another exemplary pharmaceutical composition comprises from about 15 wt% to about 70 wt % (e.g., from about 15 wt % to about 60 wt %, fromabout 15 wt % to about 50 wt %, or from about 15 wt % to about 40 wt %or from about 20 wt % to about 70 wt %, or from about 30 wt % to about70 wt %, or from about 40 wt % to about 70 wt %, or from about 50 wt %to about 70 wt %) of Compound 1 by weight of the composition, and one ormore excipients, for example, from about 20 wt % to about 50 wt % of afiller; from about 1 wt % to about 5 wt % of a disintegrant; from about2 wt % to about 0.3 wt % of a surfactant; from about 0.1 wt % to about 5wt % of a binder; from about 1 wt % to about 30 wt % of a diluent; fromabout 2 wt % to about 0.05 wt % of a glidant; and from about 2 wt % toabout 0.1 wt % of a lubricant.

Another exemplary pharmaceutical composition comprises from about 15 wt% to about 70 wt % (e.g., from about 15 wt % to about 60 wt %, fromabout 15 wt % to about 50 wt %, or from about 15 wt % to about 40 wt %or from about 20 wt % to about 70 wt %, or from about 30 wt % to about70 wt %, or from about 40 wt % to about 70 wt %, or from about 50 wt %to about 70 wt %) of Compound 1 and one or more excipients, for example,from about 20 wt % to about 50 wt % of a filler; from about 1 wt % toabout 5 wt % of a disintegrant; from about 2 wt % to about 0.3 wt % of asurfactant; from about 0.1 wt % to about 5 wt % of a binder; from about1 wt % to about 30 wt % of a diluent; from about 2 wt % to about 0.05 wt% of a glidant; and from about 2 wt % to about 0.1 wt % of a lubricant.

In one embodiment, the invention is a granular pharmaceuticalcomposition comprising:

a. about 30 wt % of Compound 1 by weight of the composition;

b. about 42 wt % of microcrystalline cellulose by weight of thecomposition;

c. about 21 wt % of mannitol by weight of the composition;

d. about 3 wt % of sodium croscarmellose sodium by weight of thecomposition;

e. about 1 wt % of sodium lauryl sulfate by weight of the composition;

f. about 2 wt % of magnesium stearate by weight of the composition; and

g. about 0.5 wt % of colloidal silica by weight of the composition.

Another granular composition formulated into an oral formulation of theinvention comprises:

a. about 50 wt % of Compound 1;

b. about 30 wt % of microcrystalline cellulose by weight of thecomposition;

c. about 13 wt % of mannitol by weight of the composition;

d. about 2 wt % of sodium croscarmellose sodium by weight of thecomposition;

e. about 4 wt % of polyvinylpyrrolidone by weight of the composition;and

f. about 1 wt % of sodium lauryl sulfate by weight of the composition.

In one embodiment, a pharmaceutical oral formulation of the inventioncomprises:

a. about 30 wt % of a Compound 1 by weight of the composition;

b. about 42 wt % of microcrystalline cellulose by weight of thecomposition;

c. about 21 wt % of mannitol by weight of the composition;

d. about 3 wt % of sodium croscarmellose sodium by weight of thecomposition;

e. about 1 wt % of sodium lauryl sulfate by weight of the composition;

f. about 2.5 wt % of magnesium stearate by weight of the composition;and

g. about 0.5 wt % of colloidal silica by weight of the composition.

Another pharmaceutical oral formulation of the invention comprises:

a. about 50 wt % of a Compound 1 by weight of the composition;

b. about 30 wt % of microcrystalline cellulose by weight of thecomposition;

c. about 13 wt % of mannitol by weight of the composition;

d. about 4 wt % of sodium croscarmellose sodium by weight of thecomposition;

e. about 4 wt % of polyvinylpyrrolidone by weight of the composition

f. about 1 wt % of sodium lauryl sulfate by weight of the composition;and

g. about 0.5 wt % of magnesium stearate by weight of the composition.

Another pharmaceutical oral formulation of the invention comprises:

a. about 60 wt % of a Compound 1 by weight of the composition;

b. about 20 wt % of microcrystalline cellulose by weight of thecomposition;

c. about 13 wt % of mannitol by weight of the composition;

d. about 4 wt % of sodium croscarmellose sodium by weight of thecomposition;

e. about 4 wt % of polyvinylpyrrolidone by weight of the composition

f. about 1 wt % of sodium lauryl sulfate by weight of the composition;and

g. about 0.5 wt % of magnesium stearate by weight of the composition.

Another pharmaceutical oral formulation of the invention comprises:

a. about 150 to 250 mg of Compound 1;

b. about 40 to 50 mg of mannitol;

c. about 120 to 130 mg of microcrystalline cellulose;

d. about 10 to 20 mg of croscarmellose sodium;

e. about 10 to 20 mg of polyvinylpyrrolidone;

f. about 1 to 5 mg of sodium lauryl sulfate; and

g. about 1 to 5 mg of magnesium stearate.

Another pharmaceutical oral formulation of the invention comprises:

a. about 200 mg of Compound 1;

b. about 43 mg of mannitol;

c. about 123 mg of microcrystalline cellulose;

d. about 15 mg of croscarmellose sodium;

e. about 13 mg of polyvinylpyrrolidone;

f. about 3 mg of sodium lauryl sulfate; and

g. about 4 mg of magnesium stearate.

Another pharmaceutical oral formulation of the invention comprises:

a. about 200 mg of Compound 1;

b. about 45 mg of mannitol;

c. about 123 mg of microcrystalline cellulose;

d. about 15 mg of croscarmellose sodium;

e. about 10.4 mg of polyvinylpyrrolidone;

f. about 2.6 mg of sodium lauryl sulfate; and

g. about 4 mg of magnesium stearate.

Another pharmaceutical oral formulation of the invention comprises:

a. about 70 wt % of a Compound 1 by weight of the composition;

b. about 12 wt % of microcrystalline cellulose by weight of thecomposition;

c. about 11 wt % of mannitol by weight of the composition;

d. about 4 wt % of sodium croscarmellose sodium by weight of thecomposition;

e. about 4 wt % of polyvinylpyrrolidone by weight of the composition

f. about 1 wt % of sodium lauryl sulfate by weight of the composition;and

g. about 0.5 wt % of magnesium stearate by weight of the composition.

The pharmaceutical compositions of the invention can be processed into atablet form, capsule form, pouch form, lozenge form, or other solid formthat is suited for oral administration. Thus in some embodiments, thepharmaceutical compositions are in tablet form.

In still another pharmaceutical oral formulation of the invention, ashaped pharmaceutical tablet composition having an initial hardness of5-21 kP ±20 percent comprises: about 30 wt % of Compound 1; about 42 wt% of microcrystalline cellulose by weight of the composition; about 21wt % of mannitol by weight of the composition; about 3 wt % of sodiumcroscarmellose sodium by weight of the composition; about 1 wt % ofsodium lauryl sulfate by weight of the composition; about 2.5 wt % ofmagnesium stearate by weight of the composition; and about 0.5 wt % ofcolloidal silica by weight of the composition. Wherein the amount ofCompound 1 in the shaped pharmaceutical tablet ranges from about 25 mgto about 250 mg, for example, 50 mg, or 75 mg, or 100 mg, or 150 mg, 200mg, or 250 mg Compound 1 per tablet.

In still another pharmaceutical oral formulation of the invention, ashaped pharmaceutical tablet composition having an initial hardness of5-21 kP ±20 percent comprises: about 49 wt % of a Compound 1; about 29wt % of microcrystalline cellulose by weight of the composition; about12.6 wt % of mannitol by weight of the composition; about 4 wt % ofsodium croscarmellose sodium by weight of the composition; about 4 wt %of polyvinylpyrrolidone by weight of the composition; about 1 wt % ofsodium lauryl sulfate by weight of the composition; and about 0.5 wt %of magnesium stearate by weight of the composition. The amount ofCompound 1 in the shaped pharmaceutical tablet ranges from about 25 mgto about 250 mg, for example, 50 mg, or 75 mg, or 100 mg, or 150 mg, 200mg, or 250 mg Compound 1 per tablet.

In certain embodiments, the shaped pharmaceutical tablet contains about100 mg of Compound 1. In certain embodiments, the shaped pharmaceuticaltablet contains about 200 mg of Compound 1.

Another aspect of the invention provides a pharmaceutical formulationconsisting of a tablet or capsule that includes a Compound 1 and otherexcipients (e.g., a filler, a disintegrant, a surfactant, a binder, aglidant, a colorant, a lubricant, or any combination thereof), each ofwhich is described above and in the Examples below, wherein the tablethas a dissolution of at least about 50% (e.g., at least about 60%, atleast about 70%, at least about 80%, at least about 90%, or at leastabout 99%) in about 30 minutes. In one example, the pharmaceuticalcomposition consists of a tablet that includes Compound 1 in an amountranging from 25 mg to 250 mg, for example, 25 mg, or 50 mg, or 75 mg, or100 mg, or 150 mg, 200 mg, or 250 mg and one or more excipients (e.g., afiller, a disintegrant, a surfactant, a binder, a glidant, a colorant, alubricant, or any combination thereof), each of which is described aboveand in the Examples below, wherein the tablet has a dissolution of fromabout 50% to about 100% (e.g., from about 55% to about 95% or from about60% to about 90%) in about 30 minutes. In another example, thepharmaceutical composition consists of a tablet that comprises acomposition comprising Compound 1; and one or more excipients from: afiller, a diluent, a disintegrant, a surfactant, a binder, a glidant,and a lubricant, wherein the tablet has a dissolution of at least about50% (e.g., at least about 60%, at least about 70%, at least about 80%,at least about 90%, or at least about 99%) in about 30 minutes.

In one embodiment, the tablet comprises a composition comprising atleast about 25 mg (e.g., at least about 30 mg, at least about 40 mg, orat least about 50 mg) of Compound 1; and one or more excipients from: afiller, a diluent, a disintegrant, a surfactant, a binder, a glidant,and a lubricant. In another embodiment, the tablet comprises acomposition comprising at least about 25 mg (e.g., at least about 30 mg,at least about 40 mg, at least about 50 mg, at least about 100 mg, or atleast 150 mg) of Compound 1 and one or more excipients from: a filler, adiluent, a disintegrant, a surfactant, a binder, a glidant, and alubricant.

Dissolution can be measured with a standard USP Type II apparatus thatemploys a dissolution media of 0.1% CTAB dissolved in 900 mL of DIwater, buffered at pH 6.8 with 50 mM potassium phosphate monoasic,stirring at about 50-75 rpm at a temperature of about 37° C. A singleexperimental tablet is tested in each test vessel of the apparatus.Dissolution can also be measured with a standard USP Type II apparatusthat employs a dissolution media of 0.7% sodium lauryl sulfate dissolvedin 900 mL of 50 mM sodium phosphate buffer (pH 6.8), stirring at about65 rpm at a temperature of about 37° C. A single experimental tablet istested in each test vessel of the apparatus. Dissolution can also bemeasured with a standard USP Type II apparatus that employs adissolution media of 0.5% sodium lauryl sulfate dissolved in 900 mL of50 mM sodium phosphate buffer (pH 6.8), stirring at about 65 rpm at atemperature of about 37° C. A single experimental tablet is tested ineach test vessel of the apparatus.

Methods for Making Compound 1, Compound 1 Form I, Compound 1 Form II,Compound 1 HCL Salt Form A Compound 1

Compound 1 is used as the starting point for the other solid state formsand can be prepared by coupling an acid chloride moiety with an aminemoiety according to Schemes 1-4.

Scheme 1 depicts the preparation of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride,which is used in Scheme 3 to make the amide linkage of Compound 1.

The starting material, 2,2-difluorobenzo[d][1,3]dioxole-5-carboxylicacid, is commercially available from Saltigo (an affiliate of theLanxess Corporation). Reduction of the carboxylc acid moiety in2,2-difluorobenzo[d][1,3]dioxole-5-carboxylic acid to the primaryalcohol, followed by conversion to the corresponding chloride usingthionyl chloride (SOCl₂), provides5-(chloromethyl)-2,2-difluorobenzo[d][1,3]dioxole, which is subsequentlyconverted to 2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetonitrile usingsodium cyanide. Treatment of2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetonitrile with base and1-bromo-2-chloroethane provides1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonitrile. Thenitrile moiety in1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonitrile isconverted to a carboxylic acid using base to give1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acid,which is converted to the desired acid chloride using thionyl chloride.

Scheme 2 depicts an alternative synthesis of the requisite acidchloride. 5-bromomethyl-2,2-difluoro-1,3-benzodioxole is coupled withethyl cyanoacetate in the presence of a palladium catalyst to form thecorresponding alpha cyano ethyl ester. Saponification of the estermoiety to the carboxylic acid gives the cyanoethyl compound. Alkylationof the cyanoethyl compound with 1-bromo-2-chloro ethane in the presenceof base gives the cyanocyclopropyl compound. Treatment of thecyanocyclopropyl compound with base gives the carboxylate salt, which isconverted to the carboxylic acid by treatment with acid. Conversion ofthe carboxylic acid to the acid chloride is then accomplished using achlorinating agent such as thionyl chloride or the like.

Scheme 3 depicts the preparation of the requisite tert-butyl3-(6-amino-3-methylpyridin-2-yl)benzoate, which is coupled with1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride inScheme 3 to give Compound 1. Palladium-catalyzed coupling of2-bromo-3-methylpyridine with 3-(tert-butoxycarbonyl)phenylboronic acidgives tert-butyl 3-(3-methylpyridin-2-yl)benzoate, which is subsequentlyconverted to the desired compound.

Scheme 4 depicts the coupling of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloridewith tert-butyl 3-(6-amino-3-methylpyridin-2-yl)benzoate using triethylamine and 4-dimethylaminopyridine to initially provide the tert-butylester of Compound 1.

Compound 1 Form I

Compound 1 Form I is prepared by dispersing or dissolving a salt form,such as the HCl salt, of Compound 1 in an appropriate solvent for aneffective amount of time. Treatment of the tert-butyl ester with an acidsuch as HCl, gives the HCL salt of Compound 1, which is typically acrystalline solid. Compound 1 Form I may also be prepared directly fromthe t-butyl ester precursor by treatment with an appropriate acid, suchas formic acid.

The HCl salt of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid can be usedto make Form I by dispersing or dissolving the HCl salt of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid in anappropriate solvent for an effective amount of time. Other salts of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid may be used,such as, for example, salts derived from other mineral or organic acids.The other salts result from acid-mediated hydrolysis of the t-butylester moiety. Salts derived from other acids may include, for example,nitric, sulfuric, phosphoric, boric, acetic, benzoic and malonic. Thesesalt forms of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid may or maynot be soluble, depending upon the solvent used, but lack of solubilitydoes not hinder formation of Form I. For example, in one embodiment, theappropriate solvent may be water or an alcohol/water mixture such as 50%methanol/water mixture, even though the HCl salt form of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid is onlysparingly soluble in water. In one embodiment, the appropriate solventis water.

The effective amount of time for formation of Form I from the salt of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid can be anytime between 2 to 24 hours or greater. It is recognized that the amountof time needed is inversely proportional to the temperature. That is,the higher the temperature the less time needed to affect dissociationof acid to form Form I. When the solvent is water, stirring thedispersion for approximately 24 hours at room temperature provides FormI in an approximately 98% yield. If a solution of the salt of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid is desiredfor process purposes, an elevated temperature may be used. Afterstirring the solution for an effective amount of time at the elevatedtemperature, recrystallization upon cooling provides substantially pureForm 1. In one embodiment, substantially pure refers to greater thanabout 90% purity. In another embodiment, substantially pure refers togreater than about 95% purity. In another embodiment, substantially purerefers to greater than about 98% purity. In another embodiment,substantially pure refers to greater than about 99% purity. Thetemperature selected depends in part on the solvent used and is wellwithin the determination capabilities of one of ordinary skill in theart. In one embodiment, the temperature is between room temperature andabout 80° C. In another embodiment, the temperature is between roomtemperature and about 40° C. In another embodiment, the temperature isbetween about 40° C. and about 60° C. In another embodiment, thetemperature is between about 60° C. and about 80° C.

Compound 1 Form I may also be formed directly from3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate (cf.Scheme 3), which is a precursor to the salt of Compound 1. Thus,3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate isallowed to undergo reaction with an appropriate acid, such as, forexample, formic acid under appropriate reaction conditions to giveCompound 1 Form I.

Compound 1 Form I may be further purified by recrystallization from anorganic solvent. Examples of organic solvents include, but are notlimited to, toluene, cumene, anisol, 1-butanol, isopropyl acetate, butylacetate, isobutyl acetate, methyl t-butyl ether, methyl isobutyl ketoneand 1-propanol-water mixtures. The temperature may be as describedabove. For example, Form I is dissolved in 1-butanol at 75° C. until itis completely dissolved. Cooling down the solution to 10° C. at a rateof 0.2° C./min yields crystals of Form I which may be isolated byfiltration.

In one embodiment, Compound 1 Form I is characterized by one or morepeaks at 15.2 to 15.6 degrees, 16.1 to 16.5 degrees, and 14.3 to 14.7degrees in an X-ray powder diffraction obtained using Cu K alpharadiation. In another embodiment, Compound 1 Form I is characterized byone or more peaks at 15.4, 16.3, and 14.5 degrees. In anotherembodiment, Compound 1 Form I is further characterized by a peak at 14.6to 15.0 degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 14.8 degrees. In another embodiment, Compound1 Form I is further characterized by a peak at 17.6 to 18.0 degrees. Inanother embodiment, Compound 1 Form I is further characterized by a peakat 17.8 degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 16.4 to 16.8 degrees. In another embodiment,Compound 1 Form I is further characterized by a peak at 16.4 to 16.8degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 16.6 degrees. In another embodiment, Compound1 Form I is further characterized by a peak at 7.6 to 8.0 degrees. Inanother embodiment, Compound 1 Form I is further characterized by a peakat 7.8 degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 25.8 to 26.2 degrees. In another embodiment,Compound 1 Form I is further characterized by a peak at 26.0 degrees. Inanother embodiment, Compound 1 Form I is further characterized by a peakat 21.4 to 21.8 degrees. In another embodiment, Compound 1 Form I isfurther characterized by a peak at 21.6 degrees. In another embodiment,Compound 1 Form I is further characterized by a peak at 23.1 to 23.5degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 23.3 degrees. In some embodiments, Compound 1Form I is characterized by a diffraction pattern substantially similarto that of FIG. 1. In some embodiments, Compound 1 Form I ischaracterized by a diffraction pattern substantially similar to that ofFIG. 2.

In some embodiments, the particle size distribution of D90 is about 82μm or less for Compound 1 Form I. In some embodiments, the particle sizedistribution of D50 is about 30 or less for Compound 1 Form I.

Compound 1 Form II

Compound 1 Form II is prepared by slurrying Compound 1 Form I in anappropriate solvent at a sufficient concentration for a sufficient time.The slurry is then filtered centrifugally or under vacuum and dried atambient conditions for sufficient time to yield Compound 1 Form

In some embodiments, about 20 to 40 mg of Compound 1 Form I is slurriedin about 400 to 600 μL of an appropriate solvent. In another embodiment,about 25 to 35 mg of Compound 1 Form I is slurried in about 450 to 550μL of an appropriate solvent. In another embodiment, about 30 mg ofCompound 1 Form I is slurried in about 500 μL of an appropriate solvent.

In some embodiments, the time that Compound 1 Form I is allowed toslurry with the solvent is froml hour to four days. More particularly,the time that Compound 1 Form I is allowed to slurry with the solvent isfrom 1 to 3 days. More particularly, the time is 2 days.

In some embodiments, the appropriate solvent is selected from an organicsolvent of sufficient size to fit the voids in the crystalline latticeof Compound 1 Form II. In other embodiments, the solvate is ofsufficient size to fit in voids measuring about 100 Å³.

In other embodiments, the solvent is selected from the group consistingof methanol, ethanol, acetone, 2-propanol, acetonitrile,tetrahydrofuran, methyl acetate, 2-butanone, ethyl formate, and 2-methyltetrahydrofuran.

In other embodiments, a mixture of two or more of these solvents may beused to obtain Compound 1 Form II. Alternatively, Compound 1 Form II maybe obtained from a mixture comprising one or more of these solvents andwater.

In some embodiments, the effective amount of time for drying Compound 1Form II is 1 to 24 hours. More particularly, the time is 6 to 18 hours.More particularly, the time is about 12 hours.

In another embodiment, Compound 1 Form II is prepared by dispersing ordissolving a salt form of Compound 1, such as an HCl salt of Compound 1in an appropriate solvent for an effective amount of time.

Compound 1 Form II as disclosed herein comprises a crystalline latticeof Compound 1 in which voids in the crystalline lattice are empty, oroccupied, or partially occupied by one or more molecules of a suitablesolvent. Suitable solvents include, but are not limited to, methanol,ethanol, acetone, 2-propanol, acetonitrile, tetrahydrofuran, methylacetate, 2-butanone, ethyl formate, and 2-methyl tetrahydrofuran.Certain physical characterisics of Compound 1 isostructural solvateforms, such as X-ray powder diffraction, melting point and DSC, are notsubstantially affected by the particular solvent molecule in question.

In one embodiment, Compound 1 Form II is characterized by one or morepeaks at 21.50 to 21.90 degrees, 8.80 to 9.20 degrees, and 10.80 to11.20 degrees in an X-ray powder diffraction obtained using Cu K alpharadiation. In another embodiment, Compound 1 Form II is characterized byone or more peaks at 21.50 to 21.90 degrees, 8.80 to 9.20 degrees, 10.80to 11.20 degrees, 18.00 to 18.40 degrees, and 22.90 to 23.30 degrees inan X-ray powder diffraction obtained using Cu K alpha radiation. Inanother embodiment, Compound 1 Form II is characterized by one or morepeaks at 21.70, 8.98, and 11.04 degrees. In another embodiment, Compound1 Form II is characterized by one or more peaks at 21.70, 8.98, 11.04,18.16, and 23.06 degrees. In another embodiment, Compound 1 Form II ischaracterized by a peak at 21.50 to 21.90 degrees. In anotherembodiment, Compound 1 Form II is further characterized by a peak at21.70 degrees. In another embodiment, Compound 1 Form II is furthercharacterized by a peak at 8.80 to 9.20 degrees. In another embodiment,Compound 1 Form II is further characterized by a peak at 8.98 degrees.In another embodiment, Compound 1 Form II is further characterized by apeak at 10.80 to 11.20 degrees. In another embodiment, Compound 1 FormII is further characterized by a peak at 11.04. In another embodiment,Compound 1 Form II is further characterized by a peak at 18.00 to 18.40degrees. In another embodiment, Compound 1 Form II is furthercharacterized by a peak at 18.16 degrees. In another embodiment,Compound 1 Form II is further characterized by a peak at 22.90 to 23.30degrees. In another embodiment, Compound 1 Form II is furthercharacterized by a peak at 23.06 degrees. In another embodiment,Compound 1 Form II is further characterized by a peak at 20.40 to 20.80degrees. In another embodiment, Compound 1 Form II is furthercharacterized by a peak at 20.63 degrees. In another embodiment,Compound 1 Form II is further characterized by a peak at 22.00 to 22.40degrees. In another embodiment, Compound 1 Form II is furthercharacterized by a peak at 22.22 degrees. In another embodiment,Compound 1 Form II is further characterized by a peak at 18.40 to 18.80degrees. In another embodiment, Compound 1 Form II is furthercharacterized by a peak at 18.57 degrees. In another embodiment,Compound 1 Form II is further characterized by a peak at 16.50 to 16.90degrees. In another embodiment, Compound 1 Form II is furthercharacterized by a peak at 16.66 degrees. In another embodiment,Compound 1 Form II is further characterized by a peak at 19.70 to 20.10degrees. In another embodiment, Compound 1 Form II is furthercharacterized by a peak at 19.86 degrees.

In some embodiments, Compound 1 Form II is characterized by adiffraction pattern substantially similar to that of FIG. 3. In someembodiments, Compound 1 Form II is characterized by diffraction patternssubstantially similar to those provided in FIG. 4.

In another embodiment, the solvate that forms Compound 1 Form II isselected from the group consisting of methanol, ethanol, acetone,2-propanol, acetonitrile, tetrahydrofuran, methyl acetate, 2-butanone,ethyl formate, and 2-methyl tetrahydrofuran. Diffraction patterns areprovided for the following Compound 1 Form II: methanol (FIG. 5),ethanol (FIG. 6), acetone (FIG. 7), 2-propanol (FIG. 8), acetonitrile(FIG. 9), tetrahydrofuran (FIG. 10), methyl acetate (FIG. 11),2-butanone (FIG. 12), ethyl formate (FIGS. 13), and2-methytetrahydrofuran (FIG. 14).

In another embodiment, the invention provides Compound 1 Form II whichexhibits two or more phase transitions as determined by DSC or a similaranalytic method known to the skilled artisan. In some embodiments, theDSC of Compound 1 Form II is substantially similar to the DSC tracedepicted in FIG. 15. In another embodiment of this aspect, the DSC givestwo phase transitions. In another embodiment, the DSC gives three phasetransitions. In another embodiment, one of the phase transitions occursbetween 200 and 207° C. In another embodiment, one of the phasetransitions occurs between 204 and 206° C. In another embodiment, one ofthe phase transitions occurs between 183 and 190° C. In anotherembodiment, one of the phase transitions occurs between 185 and 187° C.In another embodiment, the melting point of Compound 1, Solvate Form Ais between 183° C. to 190° C. In another embodiment, the melting pointof Compound 1, Solvate Form A is between 185° C. to 187° C.

In another embodiment, Compound 1 Form II comprises 1 to 10 weightpercent (wt. %) solvate as determined by TGA. In some embodiments, theTGA of Compound 1 Form II is substantially similar to the TGA tracedepicted in FIG. 16. In another embodiment, Compound 1 Form II comprises2 to 5 wt. % solvate as determined by TGA or a similar analytic methodknown to the skilled artisan.

In another embodiment, the conformation of Compound 1 Form II acetonesolvate is substantially similar to that depicted in FIG. 17, which isbased on single X-ray analysis.

In another embodiment, Compound 1 Form II acetone solvate has a P2i/nspace group, and the following unit cell dimensions:

a=16.5235 (10) Å α=90°

b=12.7425 (8) Å β=103.736 (4)°

c=20.5512 (13) Å γ=90°.

Compound 1 HCl Salt Form A

Compound 1 HCl Salt Form A can be prepared from the HCl salt of Compound1, by dissolving the HCl salt of Compound 1 in a minimum of solvent andremoving the solvent by slow evaporation. In another embodiment, thesolvent is an alcohol. In another embodiment, the solvent is ethanol.Slow evaporation is generally carried out by impeding the evaporation ofthe solvent. For example, in one embodiment, slow evaporation involvesdissolving the HCl salt of Compound 1 in a vial and covering the vialwith parafilm that contains a hole poked in it.

In one embodiment, Compound 1 HCl Salt Form A is characterized by one ormore peaks at 8.80 to 9.20 degrees, 17.30 to 17.70 degrees, and 18.20 to18.60 degrees in an X-ray powder diffraction obtained using Cu K alpharadiation. In another embodiment, Compound 1 HCl Salt Form A ischaracterized by one or more peaks at 8.80 to 9.20 degrees, 17.30 to17.70 degrees, 18.20 to 18.60 degrees, 10.10 to 10.50, and 15.80 to16.20 degrees in an X-ray powder diffraction obtained using Cu K alpharadiation. In another embodiment, Compound 1 HCl Salt Form A ischaracterized by one or more peaks at 8.96, 17.51, and 18.45 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is characterized by oneor more peaks at 8.96, 17.51, 18.45. 10.33, and 16.01 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is characterized by apeak at 8.80 to 9.20 degrees. In another embodiment, Compound 1 HCl SaltForm A is characterized by a peak at 8.96 degrees. In anotherembodiment, Compound 1 HCl Salt Form A is further characterized by apeak at 17.30 to 17.70 degrees. In another embodiment, Compound 1 HClSalt Form A is characterized by a peak at 17.51 degrees. In anotherembodiment, Compound 1 HCl Salt Form A is further characterized by apeak at 18.20 to 18.60 degrees. In another embodiment, Compound 1 HClSalt Form A is further characterized by a peak at 18.45degrees. Inanother embodiment, Compound 1 HCl Salt Form A is further characterizedby a peak at 10.10 to 10.50 degrees. In another embodiment, Compound 1HCl Salt Form A is further characterized by a peak at 10.33 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is further characterizedby a peak at 15.80 to 16.20 degrees. In another embodiment, Compound 1HCl Salt Form A is further characterized by a peak at 16.01 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is further characterizedby a peak at 11.70 to 12.10 degrees. In another embodiment, Compound 1HCl Salt Form A is further characterized by a peak at 11.94 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is further characterizedby a peak at 7.90 to 8.30 degrees. In another embodiment, Compound 1 HClSalt Form A is further characterized by a peak at 8.14 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is further characterizedby a peak at 9.90 to 10.30 degrees. In another embodiment, Compound 1HCl Salt Form A is further characterized by a peak at 10.10 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is further characterizedby a peak at 16.40 to 16.80 degrees. In another embodiment, Compound 1HCl Salt Form A is further characterized by a peak at 16.55 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is further characterizedby a peak at 9.30 to 9.70 degrees. In another embodiment, Compound 1 HClSalt Form A is further characterized by a peak at 9.54 degrees. Inanother embodiment, Compound 1 HCl Salt Form A is further characterizedby a peak at 16.40 to 16.80 degrees. In another embodiment, Compound 1HCl Salt Form A is further characterized by a peak at 16.55 degrees. Insome embodiments, Compound 1 HCl Salt Form A is characterized as a dimeras depicted in FIG. 18.

In some embodiments, Compound 1 HCl Salt Form A is characterized by adiffraction pattern substantially similar to that of FIG. 19.

In another embodiment, the invention features crystalline Compound 1 HClSalt Form A having a P⁻1 space group, and the following unit celldimensions:

a=10.2702 (2) Å α=67.0270 (10)°

b=10.8782 (2) Å β=66.1810 (10)°

c=12.4821 (3) Å γ=72.4760 (10)°.

Methods for Making the Pharmaceutical Compositions

The dosage unit forms of the invention can be produced by compacting orcompressing an admixture or composition, for example, a powder orgranules, under pressure to form a stable three-dimensional shape (e.g.,a tablet). As used herein, “tablet” includes compressed pharmaceuticaldosage unit forms of all shapes and sizes, whether coated or uncoated.

The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Ingeneral, a compacted mixture has a density greater than that of themixture prior to compaction. A dosage unit form of the invention canhave almost any shape including concave and/or convex faces, rounded orangled corners, and a rounded to rectilinear shape. In some embodiments,the compressed dosage forms of the invention comprise a rounded tablethaving flat faces. The solid pharmaceutical dosage forms of theinvention can be prepared by any compaction and compression method knownby persons of ordinary skill in the art of forming compressed solidpharmaceutical dosage forms. In particular embodiments, the formulationsprovided herein may be prepared using conventional methods known tothose skilled in the field of pharmaceutical formulation, as described,e.g., in pertinent textbooks. See, e.g., Remington: The Science andPractice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins,Baltimore, Md. (2003); Ansel et al., Pharmaceutical Dosage Forms AndDrug Delivery Systems, 7th Edition, Lippincott Williams & Wilkins,(1999); The Handbook of Pharmaceutical Excipients, 4^(th) edition, Roweet al., Eds., American Pharmaceuticals Association (2003); Gibson,Pharmaceutical Preformulation And Formulation, CRC Press (2001), thesereferences hereby incorporated herein by reference in their entirety.

Granulation and Compression

In some embodiments, solid forms, including powders comprising theactive agent Compound 1 and the included pharmaceutically acceptableexcipients (e.g. filler, diluent, disintegrant, surfactant, glidant,binder, lubricant, or any combination thereof) can be subjected to a drygranulation process. The dry granulation process causes the powder toagglomerate into larger particles having a size suitable for furtherprocessing. Dry granulation can improve the flowability of a mixture inorder to be able to produce tablets that comply with the demand of massvariation or content uniformity.

Formulations as described herein may be produced using one or moremixing and dry granulations steps. The order and the number of themixing and granulation steps do not seem to be critical. However, atleast one of the excipients and Compound 1 can be been subject to drygranulation or wet high shear granulation before compression intotablets. Dry granulation of Compound 1 and the excipients made togetherprior to tablet compression seem, surprisingly, to be a simple,inexpensive and efficient way of providing close physical contactbetween the ingredients of the present compositions and formulations andthus results in a tablet formulation with good stability properties. Drygranulation can be carried out by a mechanical process, which transfersenergy to the mixture without any use of any liquid substances (neitherin the form of aqueous solutions, solutions based on organic solutes, ormixtures thereof) in contrast to wet granulation processes, alsocontemplated herein. Generally, the mechanical process requirescompaction such as the one provided by roller compaction. An example ofan alternative method for dry granulation is slugging.

In some embodiments, roller compaction is a granulation processcomprising highly intensive mechanical compacting of one or moresubstances. In some embodiments, a pharmaceutical composition comprisingan admixture of powders is pressed, that is roller compacted, between 2counter rotating rollers to make a solid sheet which is subsequentlycrushed in a sieve to form a particulate matter. In this particulatematter, a close mechanical contact between the ingredients can beobtained. An example of roller compaction equipment is Minipactor® aGerteis 3W-Polygran from Gerteis Maschinen+Processengineering AG.

In some embodiments, tablet compression according to the invention canoccur without any use of any liquid substances (neither in the form ofaqueous solutions, solutions based on organic solutes, or mixturesthereof), i.e. a dry granulation process. In a typical embodiment theresulting core or tablet has a compressive strength in the range of 1 to15 kP; such as 1.5 to 12.5 kP, preferably in the range of 2 to 10 kP.

Brief Manufacturing Procedure

In some embodiments, the ingredients are weighed according to theformula set herein. Next, all of the intragranular ingredients aresifted and mixed well. The ingredients can be lubricated with a suitablelubricant, for example, magnesium stearate. The next step can comprisecompaction/slugging of the powder admixture and sized ingredients. Next,the compacted or slugged blends are milled into granules and sifted toobtain the desired size. Next, the granules can be further lubricatedwith, for example, magnesium stearate. Next the granular composition ofthe invention can be compressed on suitable punches into variouspharmaceutical formulations in accordance with the invention. Optionallythe tablets can be coated with a film, colorant or other coating.

Another aspect of the invention provides a method for producing apharmaceutical composition comprising providing an admixture of acomposition comprising Compound 1 and one or more excipients selectedfrom: a filler, a diluent, a binder, a glidant, a surfactant, alubricant, a disintegrant, and compressing the composition into a tablethaving a dissolution of at least about 50% in about 30 minutes.

In another embodiment, a wet granulation process is performed to yieldthe pharmaceutical formulation of the invention from an admixture ofpowdered and liquid ingredients. For example, a pharmaceuticalcomposition comprising an admixture of a composition comprising Compound1 and one or more excipients selected from: a filler, a diluent, abinder, a glidant, a surfactant, a lubricant, a disintegrant, areweighed as per the formula set herein. Next, all of the intragranularingredients are sifted and mixed in a high shear or low shear granulatoror a twin screw granulator using water or water with a surfactant orwater with a binder or water with a surfactant and a binder to granulatethe powder blend. A fluid other than water can also be used with orwithout surfactant and/or binder to granulate the powder blend. Next,the wet granules can optionally be milled using a suitable mill. Next,water may optionally be removed from the admixture by drying theingredients in any suitable manner. Next, the dried granules canoptionally be milled to the required size. Next, extra granularexcipients can be added by blending (for example a filler, a diluent,and a disintegrant). Next, the sized granules can be further lubricatedwith magnesium stearate and a disintegrant, for example, croscarmellosesodium. Next the granular composition of the invention can be compressedon suitable punches into various pharmaceutical formulations inaccordance with the invention. Optionally, the tablets can be coatedwith a film, colorant or other coating.

In a particularly favored embodiment, the pharmaceutical compositions ofthe present invention are prepared by a continuous twin screw wetgranulation (TSWG) process. Continuous manufacturing delivers highquality and highly consistent product with on-line monitoring andcontrol. Continuous manufacturing also facilitates quality by designdevelopment with a “data rich” design space and an easier to understandimpact of upstream variables on the downstream process and final productquality. In addition, the pharmaceutical compositions of the presentinvention can be finalized early on commercial scale equipment whichavoids scale-up risks and formulation changes late in development.Finally, continuous manufacturing has commercial manufacturingadvantages such as improved process control, reduced product handling,and real time release efficiencies. The overall result is a more robust,controllable, and scalable process that has fewer process checksresulting in increased product quality and therefore greater patientsafety.

For example, high shear granulation (HSG), a common granulationtechnique is well known for the risk of over-granulation and poorprocess control. Scale-up of this process is very challenging andinvolves significant risk. Changing from a HSG process to a continuousTSWG process, allows scale-up using the same equipment to producedifferent batch sizes, by running for a longer time. This eliminates thescale-up risk commonly encountered with wet granulation processes.Additionally, it was found that the TSWG process is more robust, beingless sensitive to over-granulation. As can be seen in FIG. 28 for aCompound 1 tablet, the HSG process showed significant dissolutionslow-down with increasing water content, while the TSWG process did notshow a change for a similar range of water addition. Surprisingly, noperformance changes were found with the tablet formulations comprisingCompound 1 between 45-55 percent by weight and the tablet formulationscomprising Compound 1 between 60-70 percent by weight using the twinscrew wet granulation process. This was not the case with the HSGprocess. Additionally, this continuous and increased product qualityprocess addresses a common complaint by the FDA regarding the lack ofdrug availability for patients in need thereof.

In one embodiment the continuous process starts with feeding individualexcipients and Compound 1 into a continuous in-line blender throughloss-in-weight feeding. From this blender, the material is continuouslyconveyed and processed through twin screw wet granulation, drying,milling, extra-granular excipient addition, blending, compression andfilm coating.

For example, in one embodiment, a tablet comprising Compound 1 may beprepared continuously according to the below flow chart.

Each of the ingredients of this exemplary admixture is described aboveand in the Examples below. Furthermore, the admixture can compriseoptional additives, such as, one or more colorants, one or more flavors,and/or one or more fragrances as described above and in the Examplesbelow. In some embodiments, the relative concentrations (e.g., wt %) ofeach of these ingredients (and any optional additives) in the admixtureare also presented above and in the Examples below. The ingredientsconstituting the admixture can be provided sequentially or in anycombination of additions; and, the ingredients or combination ofingredients can be provided in any order. In one embodiment, thelubricant is the last component added to the admixture.

In another embodiment, the admixture comprises a composition of Compound1, and any one or more of the excipients; a binder, a glidant, asurfactant, a diluent, a lubricant, a disintegrant, and a filler,wherein each of these ingredients is provided in a powder form (e.g.,provided as particles having a mean or average diameter, measured bylight scattering, of 250 μm or less (e.g., 150 μm or less, 100 μm orless, 50 μm or less, 45 μm or less, 40 μm or less, or 35 μm or less)).For instance, the admixture comprises a composition of Compound 1, adiluent, a glidant, a surfactant, a lubricant, a disintegrant, and afiller, wherein each of these ingredients is provided in a powder form(e.g., provided as particles having a mean diameter, measured by lightscattering, of 250 μm or less (e.g., 150 μm or less, 100 μm or less, 50μm or less, 45 μm or less, 40 μm or less, or 35 μm or less)). In anotherexample, the admixture comprises a composition of Compound 1, a diluent,a binder, a surfactant, a lubricant, a disintegrant, and a filler,wherein each of these ingredients is provided in a powder form (e.g.,provided as particles having a mean diameter, measured by lightscattering, of 250 μm or less (e.g., 150 μm or less, 100 μm or less, 50μm or less, 45 μm or less, 40 μm or less, or 35 μm or less))

In another embodiment, the admixture comprises a composition of Compound1, and any combination of: a binder, a glidant, a diluent, a surfactant,a lubricant, a disintegrant, and a filler, wherein each of theseingredients is substantially free of water. Each of the ingredientscomprises less than 5 wt % (e.g., less than 2 wt %, less than 1 wt %,less than 0.75 wt %, less than 0.5 wt %, or less than 0.25 wt %) ofwater by weight of the ingredient. For instance, the admixture comprisesa composition of Compound 1, a diluent, a glidant, a surfactant, alubricant, a disintegrant, and a filler, wherein each of theseingredients is substantially free of water. In some embodiments, each ofthe ingredients comprises less than 5 wt % (e.g., less than 2 wt %, lessthan 1 wt %, less than 0.75 wt %, less than 0.5 wt %, or less than 0.25wt %) of water by weight of the ingredient.

In another embodiment, compressing the admixture into a tablet isaccomplished by filling a form (e.g., a mold) with the admixture andapplying pressure to admixture. This can be accomplished using a diepress or other similar apparatus. In some embodiments, the admixture ofCompound 1 and excipients can be first processed into granular form. Thegranules can then be sized and compressed into tablets or formulated forencapsulation according to known methods in the pharmaceutical art. Itis also noted that the application of pressure to the admixture in theform can be repeated using the same pressure during each compression orusing different pressures during the compressions. In another example,the admixture of powdered ingredients or granules can be compressedusing a die press that applies sufficient pressure to form a tablethaving a dissolution of about 50% or more at about 30 minutes (e.g.,about 55% or more at about 30 minutes or about 60% or more at about 30minutes). For instance, the admixture is compressed using a die press toproduce a tablet hardness of at least about 5 kP (at least about 5.5 kP,at least about 6 kP, at least about 7 kP, at least about 10 kP, or atleast 15 kP). In some instances, the admixture is compressed to producea tablet hardness of between about 5 and 20 kP.

In some embodiments, tablets comprising a pharmaceutical composition asdescribed herein can be coated with about 3.0 wt % of a film coatingcomprising a colorant by weight of the tablet. In certain instances, thecolorant suspension or solution used to coat the tablets comprises about20%w/w of solids by weight of the colorant suspension or solution. Instill further instances, the coated tablets can be labeled with a logo,other image or text.

In another embodiment, the method for producing a pharmaceuticalcomposition comprises providing an admixture of a solid forms, e.g. anadmixture of powdered and/or liquid ingredients, the admixturecomprising Compound 1 and one or more excipients selected from: abinder, a glidant, a diluent, a surfactant, a lubricant, a disintegrant,and a filler; mixing the admixture until the admixture is substantiallyhomogenous, and compressing or compacting the admixture into a granularform. Then the granular composition comprising Compound 1 can becompressed into tablets or formulated into capsules as described aboveor in the Examples below. Alternatively, methods for producing apharmaceutical composition comprises providing an admixture of Compound1, and one or more excipients, e.g. a binder, a glidant, a diluent, asurfactant, a lubricant, a disintegrant, and a filler; mixing theadmixture until the admixture is substantially homogenous, andcompressing/compacting the admixture into a granular form using a rollercompactor using a dry granulation composition as set forth in theExamples below or alternatively, compressed/compacted into granulesusing a high shear wet granule compaction process as set forth in theExamples below. Pharmaceutical formulations, for example a tablet asdescribed herein, can be made using the granules prepared incorporatingCompound 1 in addition to the selected excipients described herein.

In some embodiments, the admixture is mixed by stirring, blending,shaking, or the like using hand mixing, a mixer, a blender, anycombination thereof, or the like. When ingredients or combinations ofingredients are added sequentially, mixing can occur between successiveadditions, continuously throughout the ingredient addition, after theaddition of all of the ingredients or combinations of ingredients, orany combination thereof. The admixture is mixed until it has asubstantially homogenous composition.

In another embodiment, the present invention comprises jet millingCompound 1, Compound 1 Form I, Compound 1 Form II, Compound 1 HCl SaltForm A in a suitable, conventional milling apparatus using air pressuresuitable to produce particles having a significant particle sizefraction between 0.1 microns and 50 microns. In another embodiment, theparticle size is between 0.1 microns and 20 microns. In anotherembodiment, the particles size is between 0.1 microns and 10 microns. Inanother embodiment, the particle size is between 1.0 microns and 5microns. In still another embodiment, Compound 1, Compound 1 Form I,Compound 1 Form II, Compound 1 HCl Salt Form A has a particle size D50of 2.0 microns.

In various embodiments, a second therapeutic agent can be formulatedtogether with Compound 1 to form a unitary or single dose form, forexample, a tablet or capsule.

Dosage forms prepared as above can be subjected to in vitro dissolutionevaluations according to Test 711 “Dissolution” in United StatesPharmacopoeia 29, United States Pharmacopeial Convention, Inc.,Rockville, Md., 2005 (“USP”), to determine the rate at which the activesubstance is released from the dosage forms. The content of activesubstance and the impurity levels are conveniently measured bytechniques such as high performance liquid chromatography (HPLC).

In some embodiments, the invention includes use of packaging materialssuch as containers and closures of high-density polyethylene (HDPE),low-density polyethylene (LDPE) and or polypropylene and/or glass,glassine foil, aluminum pouches, and blisters or strips composed ofaluminum or high-density polyvinyl chloride (PVC), optionally includinga desiccant, polyethylene (PE), polyvinylidene dichloride (PVDC),PVC/PE/PVDC, and the like. These package materials can be used to storethe various pharmaceutical compositions and formulations in a sterilefashion after appropriate sterilization of the package and its contentsusing chemical or physical sterilization techniques commonly employed inthe pharmaceutical arts.

Methods for Administering the Pharmaceutical Compositions

In one aspect, the pharmaceutical compositions of the invention can beadministered to a patient once daily or about every twenty four hours.Alternatively, the pharmaceutical compositions of the invention can beadministered to a patient twice daily or about every twelve hours. Thesepharmaceutical compositions are administered as oral formulationscontaining about 25 mg, 50 mg, 100 mg, 125 mg, 150 mg, 200 mg, 250 mg,or 400 mg of Compound 1. In this aspect, in addition to Compound 1, thepharmaceutical compositions comprise a filler; a diluent; adisintegrant; a surfactant; at least one of a binder and a glidant; anda lubricant. For instance, a dose of 400 mg of Compound 1, may comprisetwo tablets of the invention each containing 200 mg of Compound 1, orfour tablets of the invention each containing 100 mg of Compound 1.

It will also be appreciated that the compound and pharmaceuticallyacceptable compositions and formulations of the invention can beemployed in combination therapies; that is, Compound 1 andpharmaceutically acceptable compositions thereof can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. The particular combination oftherapies (therapeutics or procedures) to employ in a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and the desired therapeutic effect to be achieved. Itwill also be appreciated that the therapies employed may achieve adesired effect for the same disorder (for example, an inventive compoundmay be administered concurrently with another agent used to treat thesame disorder), or they may achieve different effects (e.g., control ofany adverse effects). As used herein, additional therapeutic agents thatare normally administered to treat or prevent a particular disease, forexample, a CFTR mediated disease, or condition, are known as“appropriate for the disease or condition being treated.”

In one embodiment, the additional therapeutic agent is selected from amucolytic agent, bronchodialator, an antibiotic, an anti-infectiveagent, an anti-inflammatory agent, a CFTR modulator other than Compound1 of the invention, or a nutritional agent.

In one embodiment, the additional agent is(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.In another embodiment, the additional agent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.In another embodiment, the additional agent is selected from Table 1:

TABLE 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

In another embodiment, the additional agent is any combination of theabove agents. For example, the composition may comprise Compound 1,(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide,andN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.In another example, the composition may comprise Compound 1,N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide,and any one of the compounds from Table 1, i.e. compounds 1 through 14of Table 1, or any combination thereof.

In one embodiment, the additional therapeutic agent is an antibiotic.Exemplary antibiotics useful herein include tobramycin, includingtobramycin inhaled powder (TIP), azithromycin, aztreonam, including theaerosolized form of aztreonam, amikacin, including liposomalformulations thereof, ciprofloxacin, including formulations thereofsuitable for administration by inhalation, levoflaxacin, includingaerosolized formulations thereof, and combinations of two antibiotics,e.g., fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogen phosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent is a CFTR modulator otherthan Compound 1, i.e., an agent that has the effect of modulating CFTRactivity. Exemplary such agents include ataluren (“PTC124®”;3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), sinapultide,lancovutide, depelestat (a human recombinant neutrophil elastaseinhibitor), and cobiprostone (7-{(2R, 4aR, 5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid).

In another embodiment, the additional agent is a nutritional agent.Exemplary nutritional agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

In another embodiment, the additional agent is a compound selected fromgentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat,felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMPmodulators such as rolipram, sildenafil, milrinone, tadalafil, amrinone,isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP 90inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin,lactacystin, etc.

In another embodiment, the additional agent is a compound selected from3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic acid(3,3,3-trifluoro-2-hydroxy -2-methyl-propyl)-amide;5-amino-6′-methyl-3-trifluoromethyl-[2,3]bipyridinyl-6-carboxylic acid(3,3,3-trifluoro-2-hydroxy -2-methyl-propyl)-amide;3-amino-6-cyclopropyl-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide;3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propyl)-5-(trifluoromethyl)picolinamide;3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic acid((S)-3,3,3-trifluoro-2-hydroxy -2-methyl-propyl)-amide;3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylicacid((S-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-(2,4-dichloro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylicacid ((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-(2,4-dichloro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylicacid ((R)-3,3,3-trifluoro -2-hydroxy-2-methyl-propyl)-amide;3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic acid(2-hydroxy-2-methyl-propyl)-amide;3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;(S)-3-amino-6-ethoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide;3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid((R)-3,3,3-trifluoro -2-hydroxy-2-methyl-propyl)-amide;3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic acid(3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid((S)-3,3,3-trifluoro -2-hydroxy-2-methyl-propyl)-amide;3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid((R)-3,3,3-trifluoro -2-hydroxy-2-methyl-propyl)-amide, orpharmaceutically acceptable salts thereof In another embodiment, theadditional agent is a compound disclosed in U.S. Pat. No. 8,247,436 andInternational PCT Publication WO 2011113894, each incorporated herein intheir entirety by reference.

In one embodiment, the additional agent is trimethylangelicin. Inanother embodiment, the additional agent is a compound disclosed in WO2012171954, incorporated herein in its entirety by reference.

In other embodiments, the additional agent is a compound disclosed in WO2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO2006101740. In another embodiment, the additional agent is abenzo[c]quinolizinium derivative that exhibits CFTR modulation activityor a benzopyran derivative that exhibits CFTR modulation activity. Inanother embodiment, the additional agent is a compound disclosed in U.S.Pat. No. 7,202,262, U.S. Pat. No. 6,992,096, US20060148864,US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456,WO2006044682, WO2006044505, WO2006044503, WO2006044502, or WO2004091502.In another embodiment, the additional agent is a compound disclosed inWO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006099256,WO2006127588, or WO2007044560. In another embodiment, the additionalagent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.

In one embodiment, 600 mg of Compound 1 may be administered to a subjectin need thereof followed by co-administration of 250 mg ofN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide(Compound 2). In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 600 mg of Compound 1 may be achieved by administeringthree tablets each containing 200 mg of Compound 1, four tablets eachcontaining 150 mg of Compound 1, or one table of 400 mg Compound 1 andone tablet of 200 mg Compound 1. Compound 2 may be administered as apharmaceutical composition comprising Compound 2 and a pharmaceuticallyacceptable carrier. The duration of administration may continue untilamelioration of the disease is achieved or until a subject's physicianadvises, e.g. duration of administration may be less than a week, 1week, 2 weeks, 3 weeks, or a month or longer. The co-administrationperiod may be preceded by an administration period of just Compound 1alone. For example, there could be administration of 600 mg of Compound1 for 2 weeks followed by co-administration of 250 mg of Compound 2 for1 additional week. In another embodiment, 600 mg of Compound 1 may beadministered bid (twice daily) for 28 days followed by 250 mg ofCompound 2 administered bid (twice daily) for 28 days. In anotherembodiment, 600 mg of Compound 1 may be administered qd (once a day) for28 days followed by 250 mg of Compound 2 administered qd (once a day)for 28 days. In another embodiment, 600 mg of Compound 1 may beadministered qd (once a day) for 28 days followed by co-administrationof 600 mg of Compound 1 qd (once a day) and 250 mg of Compound 2 ql2h(once every 12 hours) for 28 days. In another embodiment, 600 mg ofCompound 1 may be administered qd (once a day) and 250 mg of Compound 2administered qd (once a day).

In one embodiment, 600 mg of Compound 1 may be administered to a subjectin need thereof followed by co-administration of 450 mg ofN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide(Compound 2). In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 600 mg of Compound 1 may be achieved by administeringthree tablets each containing 200 mg of Compound 1, or four tablets eachcontaining 150 mg of Compound 1. Compound 2 may be administered as apharmaceutical composition comprising Compound 2 and a pharmaceuticallyacceptable carrier. The duration of administration may continue untilamelioration of the disease is achieved or until a subject's physicianadvises, e.g. duration of administration may be less than a week, 1week, 2 weeks, 3 weeks, or a month or longer. The co-administrationperiod may be preceded by an administration period of just Compound 1alone. For example, there could be administration of 600 mg of Compound1 for 2 weeks followed by co-administration of 450 mg of Compound 2 for1 additional week. In another embodiment, 600 mg of Compound 1 may beadministered bid (twice daily) for 28 days followed by 450 mg ofCompound 2 administered bid (twice daily) for 28 days.

In one embodiment, 400 mg of Compound 1 may be administered to a subjectin need thereof followed by co-administration of 350 mg ofN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide(Compound 2). In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 400 mg of Compound 1 may be achieved by administeringtwo tablets each containing 200 mg of Compound 1, or four tablets eachcontaining 100 mg of Compound 1. Compound 2 may be administered as apharmaceutical composition comprising Compound 2 and a pharmaceuticallyacceptable carrier. The duration of administration may continue untilamelioration of the disease is achieved or until a subject's physicianadvises, e.g. duration of administration may be less than a week, 1week, 2 weeks, 3 weeks, or a month or longer. The co-administrationperiod may be preceded by an administration period of just Compound 1alone. For example, there could be administration of 400 mg of Compound1 for 2 weeks followed by co-administration of 350 mg of Compound 2 for1 additional week. In another embodiment, 400 mg of Compound 1 may beadministered q8h (every 8 hours) for 28 days followed by 350 mg ofCompound 2 administered q8h (every 8 hours) for 28 days.

In one embodiment, 400 mg of Compound 1 may be administered to a subjectin need thereof followed by co-administration of 250 mg ofN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide(Compound 2). In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 400 mg of Compound 1 may be achieved by administeringtwo tablets each containing 200 mg of Compound 1, or four tablets eachcontaining 100 mg of Compound 1. Compound 2 may be administered as apharmaceutical composition comprising Compound 2 and a pharmaceuticallyacceptable carrier. The duration of administration may continue untilamelioration of the disease is achieved or until a subject's physicianadvises, e.g. duration of administration may be less than a week, 1week, 2 weeks, 3 weeks, or a month or longer. The co-administrationperiod may be preceded by an administration period of just Compound 1alone. For example, there could be administration of 400 mg of Compound1 for 2 weeks followed by co-administration of 150 mg or 250 mg ofCompound 2 for 1 additional week. In another embodiment, 400 mg ofCompound 1 may be administered bid (twice daily) for 28 days followed by250 mg of Compound 2 administered bid (twice daily) for 28 days. Inanother embodiment, 400 mg of Compound 1 may be administered bid (twicedaily) for 28 days followed by 250 mg of Compound 2 administered qd(once daily) for 28 days. In another embodiment, 400 mg of Compound 1may be administered qd (once a day) for 28 days followed byco-administration of 400 mg of Compound 1 qd (once a day) and 250 mg ofCompound 2 ql2h (once every 12 hours) for 28 days. In anotherembodiment, 400 mg of Compound 1 may be administered bid (twice daily)and 250 mg of Compound 2 administered qd (once daily).

In one embodiment, 400 mg of Compound 1 may be administered once a dayto a subject in need thereof followed by co-administration of 150 mg ofCompound 2 once a day. In these embodiments, the dosage amounts may beachieved by administration of one or more tablets of the invention. Forexample, administration of 400 mg of Compound 1 may be achieved byadministering two tablets each containing 200 mg of Compound 1, or fourtablets each containing 100 mg of Compound 1. Compound 2 may beadministered as a pharmaceutical composition comprising Compound 2 and apharmaceutically acceptable carrier. The duration of administration maycontinue until amelioration of the disease is achieved or until asubject's physician advises, e.g. duration of administration may be lessthan a week, 1 week, 2 weeks, 3 weeks, or a month or longer. Theco-administration period may be preceded by an administration period ofjust Compound 1 alone. For example, there could be administration of 400mg of Compound 1 for 2 weeks followed by co-administration of 150 mg or250 mg of Compound 2 for 1 additional week.

In one embodiment, 400 mg of Compound 1 may be administered once a dayto a subject in need thereof followed by co-administration of 150 mg ofCompound 2 every 12 hours. In another embodiment, 400 mg of Compound 1may be administered once a day to a subject in need thereof followed byco-administration of 250 mg of Compound 2 every 12 hours. In theseembodiments, the dosage amounts may be achieved by administration of oneor more tablets of the invention. For example, administration of 400 mgof Compound 1 may be achieved by administering two tablets eachcontaining 200 mg of Compound 1, or four tablets each containing 100 mgof Compound 1. Compound 2 may be administered as a pharmaceuticalcomposition comprising Compound 2 and a pharmaceutically acceptablecarrier. The duration of administration may continue until ameliorationof the disease is achieved or until a subject's physician advises, e.g.duration of administration may be less than a week, 1 week, 2 weeks, 3weeks, or a month or longer. The co-administration period may bepreceded by an administration period of just Compound 1 alone. Forexample, there could be administration of 400 mg of Compound 1 for 2weeks followed by co-administration of 150 mg or 250 mg of Compound 2for 1 additional week.

In another embodiment, 200 mg of Compound 1 may be administered qd (oncea day) for 28 days followed by co-administration of 200 mg of Compound 1qd (once a day) and 250 mg of Compound 2 ql2h (once every 12 hours) for28 days.

In one embodiment, the 100 mg, 200 mg, and 300 mg of Compound 1 tabletsmay be combined to form a number of different dosage amounts. Forexample, dosage amounts of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mg of Compound 1may be administered by using the 100 mg, 200 mg, and 300 mg tabletformulations and multiples thereof. For example, a dosage amount of 900mg of Compound 1 may be administered using 3 300 mg tablets ofCompound 1. A dosage amount of 600 mg of Compound 1 may be administeredusing 3 200 mg tablets of Compound 1 or 2 300 mg tablets of Compound 1.Any of the preceding dosage amounts of this paragraph my be administeredwith the amounts of Compound 2 and/or dosage schedules of the preceding3 paragraphs.

These combinations are useful for treating the diseases described hereinincluding cystic fibrosis. These combinations are also useful in thekits described herein.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

In another aspect, the invention features a kit comprising a tablet ofthe present invention, and a separate therapeutic agent orpharmaceutical composition thereof. In another embodiment, the Compound1 in the tablet is in Form I. In another embodiment, the therapeuticagent is a cystic fibrosis corrector other than Compound 1. In anotherembodiment, the therapeutic agent is a cystic fibrosis potentiator. Inanother embodiment, the therapeutic agent isN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.In another embodiment, the tablet and the therapeutic agent are inseparate containers. In another embodiment, the separate containers arebottles. In another embodiment, the separate containers are vials. Inanother embodiment, the separate containers are blister packs.

Therapeutic Uses of the Composition

In one aspect, the invention also provides a method of treating,lessening the severity of, or symptomatically treating a disease in apatient, the method comprising administering an effective amount of thepharmaceutical composition of the invention to the patient, wherein thedisease is selected from cystic fibrosis, asthma, smoke induced COPD,chronic bronchitis, rhinosinusitis, constipation, pancreatitis,pancreatic insufficiency, male infertility caused by congenitalbilateral absence of the vas deferens (CBAVD), mild pulmonary disease,idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA),liver disease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington's, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, osteoporosis, osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy, lysosomalstorage disease, Angelman syndrome, and Primary Ciliary Dyskinesia(PCD), a term for inherited disorders of the structure and/or functionof cilia, including PCD with situs inversus (also known as Kartagenersyndrome), PCD without situs inversus and ciliary aplasia.

Compound 1, as part of a combination with ivacaftor(N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide),has been granted a Breakthrough Therapy Designation from the Food andDrug Administration (FDA) for the treatment of cystic fibrosis, one ofonly two such grants at the time of the filing of this application (theother being for ivacaftor). This demonstrates a significant unmet needfor the effective treatment of the cause of cystic fibrosis oversymptomatic treatments. Additionally, a common challenge for drugsapproved by the FDA is the occasional lack of drug availability forpatients in need thereof. Accordingly, a significant unmet need existsfor the presently disclosed Compound 1 formulations and processes forpreparing them in a continuous and controlled manner.

In one aspect, the invention also provides a method of treating,lessening the severity of, or symptomatically treating a disease in apatient comprising administering an effective amount of thepharmaceutical composition of the invention to the patient, wherein thedisease is selected from generalized epilepsy with ferbrile seizuresplus (GEFS+), general epilepsy with ferbile and aferbrile seizures,myotonia, paramyotonia congenital, potassium-aggravated myotonia,hyperkalemic periodic paralysis, LQTS, LQTS/Brugada syndrome,autosomal-dominant LQTS with deafness, autosomal-recessive LQTS, LQTSwith dysmorphic features, congenital and acquired LQTS, Timothysyndrome, persistent hyperinsulinemic hypolglycemia of infancy, dilatedcardiomyopathy, autosomal-dominant LQTS, Dent disease, Osteopetrosis,Bartter syndrome type III, central core disease, malignant hyperthermia,and catecholaminergic polymorphic tachycardia.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition of the invention to the patient, whereinthe patient possesses the CFTR genetic mutation N1303K, ΔI507, or R560T.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition of the invention to the patient, whereinthe patient possesses the CFTR genetic mutation G551D. In anotherembodiment, the patient is homozygous for G551D. In another embodiment,the patient is heterozygous for G551D wherein the other CFTR geneticmutation is any one of F508del, G542X, N1303K, W1282X, R117H, R553X,1717-IG->A, 621+IG->T, 2789+5G->A, 3849+I0kbC->T, R1162X, G85E,3120+1G->A, ΔI507, I898+IG->A, 3659delC, R347P, R560T, R334W, A455E,2184delA, or 711+IG->T.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition of the invention to the patient, whereinthe patient possesses the CFTR genetic mutation F508del. In anotherembodiment, the patient is homozygous for F508del. In anotherembodiment, the patient is heterozygous for F508del wherein the otherCFTR genetic mutation is any one of G551D, G542X, N1303K, W1282X, R117H,R553X, 1717-IG->A, 621+IG->T, 2789+5G->A, 3849+10kbC->T, R1162X, G85E,3120+IG->A, ΔI507, 1898+IG->A, 3659delC, R347P, R560T, R334W, A455E,2184delA, or 711+IG->T.

In certain embodiments, the pharmaceutically acceptable compositions ofthe present invention comprising Compound 1 are useful for treating,lessening the severity of, or symptomatically treating cystic fibrosisin patients who exhibit residual CFTR activity in the apical membrane ofrespiratory and non-respiratory epithelia. The presence of residual CFTRactivity at the epithelial surface can be readily detected using methodsknown in the art, e.g., standard electrophysiological, biochemical, orhistochemical techniques. Such methods identify CFTR activity using invivo or ex vivo electrophysiological techniques, measurement of sweat orsalivary Cl⁻ concentrations, or ex vivo biochemical or histochemicaltechniques to monitor cell surface density. Using such methods, residualCFTR activity can be readily detected in patients heterozygous orhomozygous for a variety of different mutations, including patientshomozygous or heterozygous for the most common mutation, F508del, aswell as other mutations such as the G551D mutation, or the R117Hmutation. In certain embodiments, the pharmaceutical compositionscomprising Compound 1 are useful for treating, lessening the severityof, or symptomatically treating cystic fibrosis in patients who exhibitlittle to no residual CFTR activity. In certain embodiments, thepharmaceutical compositions comprising Compound 1 are useful fortreating, lessening the severity of, or symptomatically treating cysticfibrosis in patients who exhibit little to no residual CFTR activity inthe apical membrane of respiratory epithelia.

In another embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who have residual CFTR activity induced oraugmented. Such a residual CFTR inducer or augmenter can be done usingpharmacological methods. In another embodiment, the compounds andcompositions of the present invention are useful for treating orlessening the severity of cystic fibrosis in patients who have residualCFTR activity induced or augmented using or gene therapy. Such methodsincrease the amount of CFTR present at the cell surface, therebyinducing a hitherto absent CFTR activity in a patient or augmenting theexisting level of residual CFTR activity in a patient.

In one embodiment, pharmaceutical compositions of the present inventioncomprising Compound 1, as described herein, are useful for treating orlessening the severity of cystic fibrosis in patients within certaingenotypes exhibiting residual CFTR activity, e.g., Class I mutations(not synthesized), class II mutation (misfolding), class III mutations(impaired regulation or gating), class IV mutations (alteredconductance), or class V mutations (reduced synthesis).

In one embodiment, pharmaceutical compositions of the present inventioncomprising Compound 1, as described herein, are useful for treating,lessening the severity of, or symptomatically treating cystic fibrosisin patients within certain clinical phenotypes, e.g., a moderate to mildclinical phenotype that typically correlates with the amount of residualCFTR activity in the apical membrane of epithelia. Such phenotypesinclude patients exhibiting pancreatic sufficiency.

In one embodiment, pharmaceutical compositions of the present inventioncomprising Compound 1, as described herein, are useful for treating,lessening the severity of, or symptomatically treating patientsdiagnosed with pancreatic sufficiency, idiopathic pancreatitis andcongenital bilateral absence of the vas deferens, or mild lung diseasewherein the patient exhibits residual CFTR activity.

In one embodiment, pharmaceutical compositions of the present inventioncomprising Compound 1, as described herein, are useful for treating,lessening the severity of, or symptomatically treating patientsdiagnosed with pancreatic sufficiency, idiopathic pancreatitis andcongenital bilateral absence of the vas deferens, or mild lung diseasewherein the patient has wild type CFTR.

In addition to cystic fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. These include, but are not limited to, chronic obstructivepulmonary disease (COPD), dry eye disease, and Sjögren's Syndrome. COPDis characterized by airflow limitation that is progressive and not fullyreversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such ascystic fibrosis and Sjogrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjogrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Augmenters or inducers of CFTRactivity may hydrate the various organs afflicted by the disease andhelp to elevate the associated symptoms.

In one embodiment, the invention relates to a method of augmenting orinducing anion channel activity in vitro or in vivo, comprisingcontacting the channel with a pharmaceutical composition of the presentinvention. In another embodiment, the anion channel is a chloridechannel or a bicarbonate channel. In another embodiment, the anionchannel is a chloride channel.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the invention will be decided by the attendingphysician within the scope of sound medical judgment. The specificeffective dose level for any particular patient or organism will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed, and like factors wellknown in the medical arts. The term “patient”, as used herein, means ananimal, preferably a mammal, and most preferably a human.

Anywhere in the present application where a name of a compound may notcorrectly describe the structure of the compound, the structuresupersedes the name and governs.

EXAMPLES

XRPD (X-Ray Powder Diffraction)

The X-Ray diffraction (XRD) data of Compound 1, Compound 1 Form I,Compound 1 Form II, or Compound 1 HCl Salt Form A were collected on aBruker D8 DISCOVER powder diffractometer with HI-STAR 2-dimensionaldetector and a flat graphite monochromator. Cu sealed tube with Kαradiation was used at 40 kV, 35mA. The samples were placed onzero-background silicon wafers at 25° C. For each sample, two dataframes were collected at 120 seconds each at 2 different θ₂ angles: 8°and 26° . The data were integrated with GADDS software and merged withDIFFRACT^(plus)EVA software. Uncertainties for the reported peakpositions are ±0.2 degrees.

Jet Milling Description

Unmicronized Compound 1, Compound 1 Form I, Compound 1 Form II, orCompound 1 HCl Salt Form A is sieved to de-lump it prior to placing itinto the jet mill hopper. All sieves are disposable and received a wipeprior to use. Unmicronized Compound 1, Compound 1 Form I, Compound 1Form II, or Compound 1 HCl Salt Form A is added to the jet mill hopperat a controlled feeding rate using compressed nitrogen gas. The gaspressure range is 40-45/45-70 (Venturi/Mill) PSI and the feeding raterange is 0.5-1.6 Kg/Hour. The Compound 1, Compound 1 Form I, Compound 1Form II, or Compound 1 HCl Salt Form A is micronized in the mill throughparticle-particle and particle-wall collisions and the processedCompound 1, Compound 1 Form I, Compound 1 Form II, or Compound 1 HClSalt Form A is emptied into the micronized product containers. It isbelieved that one of ordinary skill in the art may also achieve Compound1, Compound 1 Form I, Compound 1 Form II, or Compound 1 HCl Salt Form Awith a favorable particle size through pin milling based in part on theconditions described above.

Differential Scanning Calorimetry (DSC)

The Differential scanning calorimetry (DSC) data of Compound 1, Compound1 Form I, Compound 1 Form II, or Compound 1 HCl Salt Form A werecollected using a DSC Q100 V9.6 Build 290 (TA Instruments, New Castle,DE). Temperature was calibrated with indium and heat capacity wascalibrated with sapphire. Samples of 3-6 mg were weighed into aluminumpans that were crimped using lids with 1 pin hole. The samples werescanned from 25° C. to 350° C. at a heating rate of 1.0° C./min and witha nitrogen gas purge of 50 ml/min. Data were collected by ThermalAdvantage Q SeriesTM version 2.2.0.248 software and analyzed byUniversal Analysis software version 4.1D (TA Instruments, New Castle,Del.). The reported numbers represent single analyses.

Compound 1 Form I, Compound 1 Form II, and Compound 1 HCl Salt Form ASingle Crystal Structure Determination

Diffraction data were acquired on Bruker Apex II diffractometer equippedwith sealed tube Cu K-alpha source and an Apex II CCD detector. Thestructure was solved and refined using SHELX program (Sheldrick, G.M.,Acta Cryst., (2008) A64, 112-122). Based on systematic absences andintensities statistics the structure was solved and refined in P2₁/nspace group.

Vitride® (sodium bis(2-methoxyethoxy)aluminum hydride [orNaAlH₂(OCH₂CH₂OCH₃)₂], 65 wgt % solution in toluene) was purchased fromAldrich Chemicals.

2,2-Difluoro-1,3-benzodioxole-5-carboxylic acid was purchased fromSaltigo (an affiliate of the Lanxess Corporation).

Preparation of (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol

Commercially available 2,2-difluoro-1,3-benzodioxole-5-carboxylic acid(1.0 eq) was slurried in toluene (10 vol). Vitride® (2 eq) was added viaaddition funnel at a rate to maintain the temperature at 15-25° C. Atthe end of the addition, the temperature was increased to 40° C. for 2hours (h), then 10% (w/w) aqueous (aq) NaOH (4.0 eq) was carefully addedvia addition funnel, maintaining the temperature at 40-50° C. Afterstirring for an additional 30 minutes (min), the layers were allowed toseparate at 40° C. The organic phase was cooled to 20° C., then washedwith water (2×1.5 vol), dried (Na₂SO₄), filtered, and concentrated toafford crude (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol that was useddirectly in the next step.

Preparation of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole

(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol (1.0 eq) was dissolved inMTBE (5 vol). A catalytic amount of 4-(N,N-dimethyl)aminopyridine (DMAP)(1 mol %) was added and SOCl₂ (1.2 eq) was added via addition funnel.The SOCl₂ was added at a rate to maintain the temperature in the reactorat 15-25° C. The temperature was increased to 30° C. for 1 h, and thenwas cooled to 20° C. Water (4 vol) was added via addition funnel whilemaintaining the temperature at less than 30° C. After stirring for anadditional 30 min, the layers were allowed to separate. The organiclayer was stirred and 10% (w/v) aq NaOH (4.4 vol) was added. Afterstirring for 15 to 20 min, the layers were allowed to separate. Theorganic phase was then dried (Na₂SO₄), filtered, and concentrated toafford crude 5-chloromethyl-2,2-difluoro-1,3-benzodioxole that was useddirectly in the next step.

Preparation of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

A solution of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole (1 eq) inDMSO (1.25 vol) was added to a slurry of NaCN (1.4 eq) in DMSO (3 vol),while maintaining the temperature between 30-40° C. The mixture wasstirred for 1 h, and then water (6 vol) was added, followed by methyltent-butyl ether (MTBE) (4 vol). After stirring for 30 min, the layerswere separated. The aqueous layer was extracted with MTBE (1.8 vol). Thecombined organic layers were washed with water (1.8 vol), dried(Na2SO4), filtered, and concentrated to afford crude(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (95%) that was useddirectly in the next step.

Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile

A reactor was purged with nitrogen and charged with 900 mL of toluene.The solvent was degassed via nitrogen sparge for no less than 16 h. Tothe reactor was then charged Na₃PO₄ (155.7 g, 949.5 mmol), followed bybis(dibenzylideneacetone) palladium (0) (7.28 g, 12.66 mmol). A 10% w/wsolution of tert-butylphosphine in hexanes (51.23 g, 25.32 mmol) wascharged over 10 min at 23° C. from a nitrogen purged addition funnel.The mixture was allowed to stir for 50 min, at which time5-bromo-2,2-difluoro-1,3-benzodioxole (75 g, 316.5 mmol) was added over1 min. After stirring for an additional 50 min, the mixture was chargedwith ethyl cyanoacetate (71.6 g, 633.0 mmol) over 5 min followed bywater (4.5 mL) in one portion. The mixture was heated to 70° C. over 40min and analyzed by HPLC every 1-2 h for the percent conversion of thereactant to the product. After complete conversion was observed(typically 100% conversion after 5-8 h), the mixture was cooled to20-25° C. and filtered through a celite pad. The celite pad was rinsedwith toluene (2×450 mL) and the combined organics were concentrated to300 mL under vacuum at 60-65° C. The concentrate was charged with 225mLDMSO and concentrated under vacuum at 70-80° C. until activedistillation of the solvent ceased. The solution was cooled to 20-25° C.and diluted to 900 mL with DMSO in preparation for Step 2. ¹H NMR (500MHz, CDCl₃) δ 7.16-7.10 (m, 2H), 7.03 (d, J=8.2 Hz, 1H), 4.63 (s, 1H),4.19 (m, 2H), 1.23 (t, J=7.1 Hz, 3H).

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

The DMSO solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile fromabove was charged with 3 N HCl (617.3 mL, 1.85 mol) over 20 min whilemaintaining an internal temperature <40° C. The mixture was then heatedto 75° C. over 1 h and analyzed by HPLC every 1-2 h for % conversion.When a conversion of >99% was observed (typically after 5-6 h), thereaction was cooled to 20-25° C. and extracted with MTBE (2×525 mL),with sufficient time to allow for complete phase separation during theextractions. The combined organic extracts were washed with 5% NaCl(2×375 mL). The solution was then transferred to equipment appropriatefor a 1.5-2.5 Torr vacuum distillation that was equipped with a cooledreceiver flask. The solution was concentrated under vacuum at <60° C. toremove the solvents. (2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrilewas then distilled from the resulting oil at 125-130° C. (oventemperature) and 1.5-2.0 Torr.(2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrile was isolated as a clearoil in 66% yield from 5-bromo-2,2-difluoro-1,3-benzodioxole (2 steps)and with an HPLC purity of 91.5% AUC (corresponds to a w/w assay of95%). ¹H NMR (500 MHz, DMSO) δ 7.44 (br s, 1H), 7.43 (d, J=8.4 Hz, 1H),7.22 (dd, J=8.2, 1.8 Hz, 1H), 4.07 (s, 2H).

Preparation of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile

A mixture of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (1.0 eq),50 wt % aqueous KOH (5.0 eq) 1-bromo-2-chloroethane (1.5 eq), andOct₄NBr (0.02 eq) was heated at 70° C. for 1 h. The reaction mixture wascooled, then worked up with MTBE and water. The organic phase was washedwith water and brine. The solvent was removed to afford(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile.

Preparation of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid

(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile washydrolyzed using 6 M NaOH (8 equiv) in ethanol (5 vol) at 80° C.overnight. The mixture was cooled to room temperature and the ethanolwas evaporated under vacuum. The residue was taken up in water and MTBE,1 M HCl was added, and the layers were separated. The MTBE layer wasthen treated with dicyclohexylamine (DCHA) (0.97 equiv). The slurry wascooled to 0° C., filtered and washed with heptane to give thecorresponding DCHA salt. The salt was taken into MTBE and 10% citricacid and stirred until all the solids had dissolved. The layers wereseparated and the MTBE layer was washed with water and brine. A solventswap to heptane followed by filtration gave1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid afterdrying in a vacuum oven at 50° C. overnight.

Preparation of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonyl chloride

1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid (1.2eq) is slurried in toluene (2.5 vol) and the mixture was heated to 60°C. SOCl₂ (1.4 eq) was added via addition funnel. The toluene and SOCl₂were distilled from the reaction mixture after 30 minutes. Additionaltoluene (2.5 vol) was added and the resulting mixture was distilledagain, leaving the product acid chloride as an oil, which was usedwithout further purification.

Preparation of tert-butyl-3-(3-methylpyridin-2-yl)benzoate.

2-Bromo-3-methylpyridine (1.0 eq) was dissolved in toluene (12 vol).K₂CO₃ (4.8 eq) was added, followed by water (3.5 vol). The resultingmixture was heated to 65° C. under a stream of N₂ for 1 hour.3-(t-Butoxycarbonyl)phenylboronic acid (1.05 eq) and Pd(dppf)Cl₂.CH₂Cl₂(0.015 eq) were then added and the mixture was heated to 80° C. After 2hours, the heat was turned off, water was added (3.5 vol), and thelayers were allowed to separate. The organic phase was then washed withwater (3.5 vol) and extracted with 10% aqueous methanesulfonic acid (2eq MsOH, 7.7 vol). The aqueous phase was made basic with 50% aqueousNaOH (2 eq) and extracted with EtOAc (8 vol). The organic layer wasconcentrated to afford crude tent-butyl-3-(3-methylpyridin-2-yl)benzoate(82%) that was used directly in the next step.

Preparation of2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide

tert-Butyl-3-(3-methylpyridin-2-yl)benzoate (1.0 eq) was dissolved inEtOAc (6 vol). Water (0. 3 vol) was added, followed by urea-hydrogenperoxide (3 eq). Phthalic anhydride (3 eq) was then added portionwise tothe mixture as a solid at a rate to maintain the temperature in thereactor below 45° C. After completion of the phthalic anhydrideaddition, the mixture was heated to 45° C. After stirring for anadditional 4 hours, the heat was turned off 10% w/w aqueous Na₂SO₃ (1.5eq) was added via addition funnel. After completion of Na₂SO₃ addition,the mixture was stirred for an additional 30 min and the layersseparated. The organic layer was stirred and 10% wt/wt aqueous. Na₂CO₃(2 eq) was added. After stirring for 30 minutes, the layers were allowedto separate. The organic phase was washed 13% w/v aq NaCl. The organicphase was then filtered and concentrated to afford crude2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide (95%) thatwas used directly in the next step.

Preparation of tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate

A solution of 2-(3-(tent-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide(1 eq) and pyridine (4 eq) in acetonitrile (8 vol) was heated to 70° C.A solution of methanesulfonic anhydride (1.5 eq) in MeCN (2 vol) wasadded over 50 min via addition funnel while maintaining the temperatureat less than 75° C. The mixture was stirred for an additional 0.5 hoursafter complete addition. The mixture was then allowed to cool toambient. Ethanolamine (10 eq) was added via addition funnel. Afterstirring for 2 hours, water (6 vol) was added and the mixture was cooledto 10° C. After stirring for 3 hours, the solid was collected byfiltration and washed with water (3 vol), 2:1 acetonitrile/water (3vol), and acetonitrile (2×1.5 vol). The solid was dried to constantweight (<1% difference) in a vacuum oven at 50° C. with a slight N₂bleed to afford tent-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate as ared-yellow solid (53% yield).

Preparation of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate

The crude acid chloride described above was dissolved in toluene (2.5vol based on acid chloride) and added via addition funnel to a mixtureof tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate (1 eq), DMAP,(0.02 eq), and triethylamine (3.0 eq) in toluene (4 vol based ontent-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate). After 2 hours,water (4 vol based ontent-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate) was added to thereaction mixture. After stirring for 30 minutes, the layers wereseparated. The organic phase was then filtered and concentrated toafford a thick oil of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate(quantitative crude yield). Acetonitrile (3 vol based on crude product)was added and distilled until crystallization occurs. Water (2 vol basedon crude product) was added and the mixture stirred for 2 h. The solidwas collected by filtration, washed with 1:1 (by volume)acetonitrile/water (2×1 volumes based on crude product), and partiallydried on the filter under vacuum. The solid was dried to a constantweight (<1% difference) in a vacuum oven at 60° C. with a slight N₂bleed to afford 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate as abrown solid.

Preparation of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxo1-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.HCL salt

To a slurry of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate (1.0 eq)in MeCN (3.0 vol) was added water (0.83 vol) followed by concentratedaqueous HCl (0.83 vol). The mixture was heated to 45±5° C. Afterstirring for 24 to 48 h, the reaction was complete, and the mixture wasallowed to cool to ambient. Water (1.33 vol) was added and the mixturestirred. The solid was collected by filtration, washed with water (2×0.3vol), and partially dried on the filter under vacuum. The solid wasdried to a constant weight (<1% difference) in a vacuum oven at 60° C.with a slight N₂ bleed to afford3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.HCl as anoff-white solid.

An ¹HNMR spectrum of Compound 1 is shown in FIG. 20 and FIG. 21 depictsan ¹HNMR spectrum of Compound 1 as an HCl salt.

Table 2 below recites the ¹NHMR data for Compound I.

TABLE 2 Compound LC/MS LC/RT No M + 1 minutes NMR 1 453.3 1.93 ¹HNMR(400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99-7.93 (m, 3H), 7.80-7.78 (m, 1H),7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m, 2H), 2.24 (s, 3H),1.53-1.51 (m, 2H), 1.19-1.17 (m, 2H).

Preparation of Compound 1 Form I, Method A

A slurry of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.HCl (1 eq) inwater (10 vol) was stirred at ambient temperature. A sample was takenafter stirring for 24 h. The sample was filtered and the solid waswashed with water (2 times). The solid sample was submitted for DSCanalysis. When DSC analysis indicated complete conversion to Form I, thesolid was collected by filtration, washed with water (2×1.0 vol), andpartially dried on a filter under vacuum. The solid was then5 dried to aconstant weight (<1% difference) in a vacuum oven at 60° C. with aslight N₂ bleed to afford Compound 1 Form I as an off-white solid (98%yield). ¹H NMR (400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99-7.93 (m, 3H),7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m,2H), 2.24 (s, 3H), 1.53-1.51 (m, 2H), 1.19-1.17 (m, 2H).

Preparation of Compound 1 Form I, Method B

A solution of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate (1.0 eq)in formic acid (3.0 vol) was heated with stirring to 70±10° C. , for 8h. The reaction was deemed complete when no more than 1.0% AUC bychromatographic methods of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate)remained. The mixture was allowed to cool to ambient. The solution wasadded to water (6 vol), heated at 50° C., and the mixture was stirred.The mixture was then heated to 70±10° C. until the level of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate was nomore than 0.8% (AUC). The solid was collected by filtration, washed withwater (2×3 vol), and partially dried on the filter under vacuum. Thesolid was dried to a constant weight (<1% difference) in a vacuum ovenat 60° C. with a slight N₂ bleed to afford Compound 1 Form I as anoff-white solid.

The DSC trace of Compound 1 Form I is shown in FIG. 22. Melting forCompound 1 Form I occurs at about 204° C.

An X-ray diffraction pattern was calculated from a single crystalstructure of Compound 1 Form I and is shown in FIG. 1. Table 3 lists thecalculated peaks for FIG. 1.

TABLE 3 2θ Angle Relative Peak Rank [degrees] Intensity [%] 11 14.4148.2 8 14.64 58.8 1 15.23 100.0 2 16.11 94.7 3 17.67 81.9 7 19.32 61.3 421.67 76.5 5 23.40 68.7 9 23.99 50.8 6 26.10 67.4 10 28.54 50.1

An actual X-ray powder diffraction pattern of Compound 1 Form I is shownin FIG. 2. Table 4 lists the actual peaks for FIG. 2.

TABLE 4 2θ Angle Relative Peak Rank [degrees] Intensity [%] 7 7.83 37.73 14.51 74.9 4 14.78 73.5 1 15.39 100.0 2 16.26 75.6 6 16.62 42.6 517.81 70.9 9 21.59 36.6 10 23.32 34.8 11 24.93 26.4 8 25.99 36.9

Colorless crystals of Compound 1 Form I were obtained by cooling aconcentrated 1-butanol solution from 75° C. to 10° C. at a rate of 0.2°C./min. A crystal with dimensions of 0.50×0.08×0.03 mm was selected,cleaned with mineral oil, mounted on a MicroMount and centered on aBruker APEXII system. Three batches of 40 frames separated in reciprocalspace were obtained to provide an orientation matrix and initial cellparameters. Final cell parameters were obtained and refined based on thefull data set.

A diffraction data set of reciprocal space was obtained to a resolutionof 0.82 Å using 0.5° steps using 30 s exposure for each frame. Data werecollected at 100 (2) K. Integration of intensities and refinement ofcell parameters were accomplished using APEXII software. Observation ofthe crystal after data collection showed no signs of decomposition.

A conformational picture of Compound 1 Form I based on single crystalX-ray analysis is shown in FIG. 23. Compound 1 Form I is monoclinic,P₂1/n, with the following unit cell dimensions: a=4.9626(7) Å,b=12.299(2) Å, c=33.075 (4) Å, β=93.938(9)°, V=2014.0 Å³, Z=4. Densityof Compound 1 Form I calculated from structural data is 1.492 g/cm³ at100 K.

Preparation of Compound 1 Form II from Compound 1 Form I.

Compound 1 Form I (approximately 30 mg) was slurried in 500 μL of anappropriate solvent (for example, methanol, ethanol, acetone,2-propanol, acetonitrile, tetrahydrofuran, methyl acetate, 2-butanone,ethyl formate, and -methyl tetrahydrofuran for two days. The slurry wasthen filitered centrifugally or under vacuum and was left to dry atambient temperature overnight to yield Compound 1 Form II.

The DSC trace of Compound 1 Form II Acetone Solvate is shown in FIG. 15,showing two phase transitions. The melting point for Compound 1 Form IIAcetone Solvate occurs at about 188° C. and 205° C.

An actual X-ray powder diffraction pattern of Compound 1 Form II isshown in FIG. 3. Table 5 lists the actual peaks for FIG. 3 in descendingorder of relative intensity.

TABLE 5 2θ Angle Relative [degrees] Intensity [%] 21.70 100.0 8.98 65.511.04 57.4 18.16 55.9 23.06 55.4 20.63 53.1 22.22 50.2 18.57 49.1 16.6647.2 19.86 35.0

Conformational depictions of Compound 1 Form II Acetone Solvate based onsingle crystal X-ray analysis are shown in FIG. 24. The stoichiometrybetween Compound 1 Form II and acetone is approximately 4.4:1 (4.48:1calculated from ¹H NMR; 4.38:1 from X-ray). The crystal structurereveals a packing of the molecules where there are two voids or pocketsper unit cell, or 1 void per host molecule. In the acetone solvate,approximately 92 percent of voids are occupied by acetone molecules.Compound 1 Form II is a monoclinic P2₁/n space group with the followingunit cell dimensions: a=16.5235(10) Å, b=12.7425(8) Å, c=20.5512 (13) Å,α=90°, β=103.736(4)°, γ=90°, V=4203.3(5) Å³,=4. The density of Compound1 in Compound 1 Form II calculated from structural data is 1.430/cm³ at100 K.

A solid state ¹³C NMR spectrum of Compound 1 Form II Acetone Solvate isshown in FIG. 25. Table 6 provides chemical shifts of the relevantpeaks.

TABLE 6 Compound 1 Form II, Acetone Solvate ¹³C Chem. Shifts Peak # F1[ppm] Intensity 1 202.8 6.05 2 173.3 62.66 3 171.9 20.53 4 153.5 28.41 5150.9 21.68 6 150.1 19.49 7 143.2 45.74 8 142.3 42.68 9 140.1 37.16 10136.6 26.82 11 135.9 30.1 12 134.6 39.39 13 133.2 23.18 14 131.0 60.9215 128.5 84.58 16 116.0 34.64 17 114.2 23.85 18 112.4 25.3 19 110.924.12 20 107.8 18.21 21 32.0 54.41 22 22.2 20.78 23 18.8 100

A solid state ¹⁹F NMR spectrum of Compound 1 Form II Acetone Solvate isshown in FIG. 26. Peaks with an asterisk denote spinning side bands.Table 7 provides chemical shifts of the relevant peaks.

TABLE 7 Compound 1 Form II, Acetone Solvate ¹⁹F Chem. Shifts Peak # F1[ppm] Intensity 1 −41.6 12.5 2 −46.4 6.77 3 −51.4 9.05

Preparation of Compound 1 HCl Salt Form A.

Colorless crystals of Compound 1 HCl Salt Form A were obtained by slowevaporation from a concentrated solution of the HCl salt of Compound 1in ethanol. A crystal with dimensions of 0.30×1/5×0.15 mm was selected,cleaned using mineral oil, mounted on a MicroMount and centered on aBruker APEXII diffractometer. Three batches of 40 frames separated inreciprocal space were obtained to provide an orientation matrix andinitial cell parameters. Final cell parameters were obtained and refinedbased on the full data set.

FIG. 18 provides a conformational image of Compound 1 HCl Salt Form A asa dimer, based on single crystal analysis. An X-ray diffraction patternof Compound 1 HCl Salt Form A calculated from the crystal structure isshown in FIG. 27. Table 8 contains the calculated peaks for FIG. 27 indescending order of relative intensity.

TABLE 8 2θ Relative [degrees] Intensity [%] 8.96 100.00 17.51 48.2018.45 34.60 10.33 32.10 16.01 18.90 11.94 18.40 8.14 16.20 10.10 13.9016.55 13.30 9.54 10.10 16.55 13.30

Exemplary Oral Pharmaceutical Formulations Comprising Compound 1

A tablet was prepared with the components and amounts listed in Table 9for Exemplary Tablet 1A comprising 100mg of API, i.e. Compound 1 Form I.Exemplary Tablet 1A (formulated to have 100 mg of Compound 1) isprepared using a dry roller compaction device formulation process. InTable 9, grades/brands were microcrystalline cellulose: Avicel PH102;mannitol: Pearlitol SD 100; croscarmellose sodium: Acdisol; andcolloidal silica: Cabosil.

TABLE 9 (% w/w) Roller Compaction Granule Blend Compound 1 Form I 30Microcrystalline cellulose 42.3 Mannitol 21.2 Croscarmellose Sodium 3Sodium Lauryl Sulfate 1 Colloidal Silica 0.5 Magnesium Stearate 2 TabletComposition (100 mg dose, 335 mg image) Roller Compaction Granule Blend99.5 Magnesium Stearate 0.5

A tablet was prepared with the components and amounts listed in Table 10for Exemplary Tablet 1B comprising 100mg of API, i.e. Compound 1 Form I.Exemplary Tablet 1B (formulated to have 100 mg of Compound 1 Form I) isprepared using a wet high shear granule formulation process. In Table10, grades/brands were as follows. High Shear GranuleBlend—microcrystalline cellulose: Avicel PH101; mannitol: Pearlitol C50;croscarmellose sodium: Acdisol; polyvinylpyrrolidone: Kollidon PVP K30;and in the Tablet Composition—croscarmellose sodium: Acdisol.

TABLE 10 (% w/w) High Shear Granule Blend Compound 1 Form I 50Microcrystalline cellulose 30 Mannitol 13 Croscarmellose Sodium 2Polyvinylpyrrolidone 4 Sodium Lauryl Sulfate 1 Tablet Composition (l00mg dose, 205 mg image) High Shear Granule Blend 97.5 CroscarmelloseSodium 2.0 Magnesium Stearate 0.5

A tablet was prepared with the components and amounts listed in Table 11for Exemplary Tablet 1C comprising 100 mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1C (formulated to have 100 mg ofcrystalline Compound 1 Form I) is prepared using a wet high sheargranule formulation process. In Table 11, grades/brands were as follows.High Shear Granule Blend—microcrystalline cellulose: Avicel PH101;mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the TabletComposition—croscarmellose sodium: Acdisol.

TABLE 11 (% w/w) High Shear Granule Blend Compound 1 Form I 60Microcrystalline cellulose 20 Mannitol 13 Croscarmellose Sodium 2Polyvinylpyrrolidone 4 Sodium Lauryl Sulfate 1 Tablet Composition (l00mg dose, 171 mg image) High Shear Granule Blend 97.5 CroscarmelloseSodium 2.0 Magnesium Stearate 0.5

A tablet was prepared with the components and amounts listed in Table 12for Exemplary Tablet 1D comprising 200mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1D (formulated to have 200 mg ofcrystalline Compound 1 Form I) is prepared using a wet high sheargranule formulation process. In Table 12, grades/brands were as follows.High Shear Granule Blend—microcrystalline cellulose: Avicel PH101;mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the TabletComposition—microcrystalline cellulose: Avicel PH200; croscarmellosesodium: Acdisol; and magnesium stearate: 5712.

TABLE 12 (% w/w) High Shear Granule Blend Compound 1 Form I 60Microcrystalline cellulose 20 Mannitol 13 Croscarmellose Sodium 2Polyvinylpyrrolidone 4 Sodium Lauryl Sulfate 1 Tablet Composition (200mg dose, 402 mg image) High Shear Granule Blend 83 Microcrystallinecellulose 14 Croscarmellose Sodium 2 Magnesium Stearate 1

A tablet was prepared with the components and amounts listed in Table 13for Exemplary Tablet 1E comprising 200 mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1E (formulated to have 200 mg ofcrystalline Compound 1 Form I) is prepared using a wet high sheargranule formulation process. In Table 13, grades/brands were as follows.High Shear Granule Blend—microcrystalline cellulose: Avicel PH101;mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the Core TabletComposition—microcrystalline cellulose: Avicel PH200; croscarmellosesodium: Acdisol; and magnesium stearate: 5712; and in the film coat—filmcoat: Opadry II; wax: Carnauba.

TABLE 13 mg High Shear Granule Blend Compound 1 Form I 200Microcrystalline cellulose 66 Mannitol 43 Croscarmellose Sodium 7Polyvinylpyrrolidone 13 Sodium Lauryl Sulfate 3 Core Tablet Composition(200 mg dose, 400 mg image) High Shear Granule Blend 332Microcrystalline cellulose 56 Croscarmellose Sodium 8 Magnesium Stearate4 Film Coated Tablet (200 mg dose, 412 mg image) Core Tablet Composition400 Film Coat 12 Wax trace

A tablet was prepared with the components and amounts listed in Table 14for Exemplary Tablet 1F comprising 200 mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1F (formulated to have 200 mg ofcrystalline Compound 1 Form I) is prepared using a wet high sheargranule formulation process. In Table 14, grades/brands were as follows.High Shear Granule Blend—microcrystalline cellulose: Avicel PH101;mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the Core TabletComposition—microcrystalline cellulose: Avicel PH200; croscarmellosesodium: Acdisol; and magnesium stearate: 5712; and in the film coat—filmcoat: Opadry II; wax: Carnauba.

TABLE 14 mg High Shear Granule Blend Compound 1 Form I 200Microcrystalline cellulose 67 Mannitol 45 Croscarmellose Sodium 7Polyvinylpyrrolidone 10.4 Sodium Lauryl Sulfate 2.6 Core TabletComposition (200 mg dose, 400 mg image) High Shear Granule Blend 332Microcrystalline cellulose 56 Croscarmellose Sodium 8 Magnesium Stearate4 Film Coated Tablet (200 mg dose, 412 mg image) Core Tablet Composition400 Film Coat 12 Wax 0.04

A tablet was prepared with the components and amounts listed in Table 15for Exemplary Tablet 1G comprising 100 mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1G (formulated to have 100 mg ofcrystalline Compound 1 Form I) is prepared using a wet high sheargranule formulation process. In Table 15, grades/brands were as follows.High Shear Granule Blend - microcrystalline cellulose: Avicel PH101;mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the TabletComposition—croscarmellose sodium: Acdisol.

TABLE 15 (% w/w) High Shear Granule Blend Compound 1 Form I 70Microcrystalline cellulose 12 Mannitol 11 Croscarmellose Sodium 2Polyvinylpyrrolidone 4 Sodium Lauryl Sulfate 1 Tablet Composition (l00mg dose, 147 mg image) High Shear Granule Blend 97.5 CroscarmelloseSodium 2.0 Magnesium Stearate 0.5

A tablet was prepared with the components and amounts listed in Table 16for Exemplary Tablet 1H comprising 100 mg of API, i.e. crystallineCompound 1 Form I or Form II. Exemplary Tablet 1H (formulated to have100 mg of crystalline Compound 1 Form I or Form II) is prepared using awet high shear granule formulation process. In Table 16, grades/brandswere as follows. High Shear Granule Blend—microcrystalline cellulose:Avicel PH101; mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the Core TabletComposition—microcrystalline cellulose: Avicel PH200; croscarmellosesodium: Acdisol; and magnesium stearate: 5712.

TABLE 16 (% w/w) High Shear Granule Blend Compound 1 Form I or Form II61 Microcrystalline cellulose 20.3 Mannitol 13.2 Croscarmellose Sodium 2Polyvinylpyrrolidone 2.7 Sodium Lauryl Sulfate 0.7 Tablet Composition(100 mg dose, 197 mg image) High Shear Granule Blend 83 Microcrystallinecellulose 14 Croscarmellose Sodium 2 Magnesium Stearate 1

A tablet was prepared with the components and amounts listed in Table 17for Exemplary Tablet 1I comprising 100 mg of API, i.e. crystallineCompound 1 Form I or Form II. Exemplary Tablet 1I (formulated to have100 mg of crystalline Compound 1 Form I or Form II) is prepared using awet high shear granule formulation process. In Table 17, grades/brandswere as follows. High Shear Granule Blend—microcrystalline cellulose:Avicel PH101; mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the Core TabletComposition—microcrystalline cellulose: Avicel PH200; croscarmellosesodium: Acdisol; and magnesium stearate: 5712.

TABLE 17 mg High Shear Granule Blend Compound 1 Form I or Form II 100Microcrystalline cellulose 33.3 Mannitol 21.7 Croscarmellose Sodium 3.3Polyvinylpyrrolidone 4.4 Sodium Lauryl Sulfate 1.1 Core TabletComposition (100 mg dose, 197 mg image) High Shear Granule Blend 163.9Microcrystalline cellulose 27.6 Croscarmellose Sodium 3.9 MagnesiumStearate 2.0

A tablet was prepared with the components and amounts listed in Table 18for Exemplary Tablet 1J comprising 300 mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1J (formulated to have 300 mg ofcrystalline Compound 1 Form I) is prepared using a wet high sheargranule formulation process. In Table 18, grades/brands were as follows.High Shear Granule Blend—microcrystalline cellulose: Avicel PH101;mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the Core TabletComposition—microcrystalline cellulose: Avicel PH200; croscarmellosesodium: Acdisol; and magnesium stearate: 5712; and in the film coat—filmcoat: Opadry II; wax: Carnauba.

TABLE 18 mg High Shear Granule Blend Compound 1 Form I 300Microcrystalline cellulose 99 Mannitol 64.5 Croscarmellose Sodium 10.5Polyvinylpyrrolidone 19.5 Sodium Lauryl Sulfate 4.5 Core TabletComposition (300 mg dose, 600 mg image) High Shear Granule Blend 498Microcrystalline cellulose 84 Croscarmellose Sodium 12 MagnesiumStearate 6 Film Coated Tablet (300 mg dose, 618 mg image) Core TabletComposition 600 Film Coat 18 Wax 0.06

A tablet was prepared with the components and amounts listed in Table 19for Exemplary Tablet 1K comprising 300 mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1K (formulated to have 300 mg ofcrystalline Compound 1 Form I) is prepared using a wet high sheargranule formulation process. In Table 19, grades/brands were as follows.High Shear Granule Blend—microcrystalline cellulose: Avicel PH101;mannitol: Pearlitol C50; croscarmellose sodium: Acdisol;polyvinylpyrrolidone: Kollidon PVP K30; and in the Core TabletComposition—microcrystalline cellulose: Avicel PH200; croscarmellosesodium: Acdisol; and magnesium stearate: 5712; and in the film coat—filmcoat: Opadry II; wax: Carnauba.

TABLE 19 mg High Shear Granule Blend Compound 1 Form I 300Microcrystalline cellulose 100.5 Mannitol 67.5 Croscarmellose Sodium10.5 Polyvinylpyrrolidone 15.6 Sodium Lauryl Sulfate 3.9 Core TabletComposition (300 mg dose, 600 mg image) High Shear Granule Blend 498Microcrystalline cellulose 84 Croscarmellose Sodium 12 MagnesiumStearate 6 Film Coated Tablet (300 mg dose, 618 mg image) Core TabletComposition 600 Film Coat 18 Wax 0.06

A tablet was prepared with the components and amounts listed in Table 20for Exemplary Tablet 1L comprising 200 mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1L (formulated to have 200 mg ofcrystalline Compound 1 Form I) is prepared using a twin screw wetgranulation formulation process. In Table 20, grades/brands were asfollows. Twin Screw Granule Blend - microcrystalline cellulose: AvicelPH101; croscarmellose sodium: Acdisol; polyvinylpyrrolidone: KollidonPVP K30; and in the Core Tablet Composition—microcrystalline cellulose:Avicel PH200; croscarmellose sodium: Acdisol; and magnesium stearate:5712; and in the film coat—film coat: Opadry II; wax: Carnauba.

TABLE 20 mg Twin Screw Granule Blend Compound 1 Form I 200Microcrystalline cellulose 34.0 Croscarmellose Sodium 6.3Polyvinylpyrrolidone 7.8 Sodium Lauryl Sulfate 1.8 Core TabletComposition (200 mg dose) Twin Screw Granule Blend 249.9Microcrystalline cellulose 36.1 Croscarmellose Sodium 12.0 MagnesiumStearate 3.0 Film Coated Tablet (200 mg dose, 310 mg total) Core TabletComposition 301 Film Coat 9.0 Wax trace

A tablet was prepared with the components and amounts listed in Table 21for Exemplary Tablet 1M comprising 400 mg of API, i.e. crystallineCompound 1 Form I. Exemplary Tablet 1M (formulated to have 400 mg ofcrystalline Compound 1 Form I) is prepared using a twin screw wetgranule formulation process. In Table 21, grades/brands were as follows.Twin Screw Granule Blend—microcrystalline cellulose: Avicel PH101;croscarmellose sodium: Acdisol; polyvinylpyrrolidone: Kollidon PVP K30;and in the Core Tablet Composition—microcrystalline cellulose: AvicelPH200; croscarmellose sodium: Acdisol; and magnesium stearate: 5712; andin the film coat—film coat: Opadry II; wax: Carnauba.

TABLE 21 mg Twin Screw Granule Blend Compound 1 Form I 400Microcrystalline cellulose 68.0 Croscarmellose Sodium 12.6Polyvinylpyrrolidone 15.6 Sodium Lauryl Sulfate 3.6 Core TabletComposition (400 mg dose) Twin Screw Granule Blend 499.8Microcrystalline cellulose 72.2 Croscarmellose Sodium 24.0 MagnesiumStearate 6.0 Film Coated Tablet (400 mg dose, 620 mg total) Core TabletComposition 602 Film Coat 18.0 Wax trace

Tablet Formation from Roller Compaction Granule Composition

Equipment/Process

Equipment

Roller Compactors: Alexanderwerk WP 120, Vector TF-Mini, or VectorTF-Labo.

Screening/Weighing

Compound 1 and excipients may be screened prior to or after weigh-out.Appropriate screen sizes are mesh 20, mesh 40, or mesh 60. Compound 1may be pre-blended with one or more of the excipients to simplifyscreening.

Blending

Compound 1 and excipients may be added to the blender in differentorder. The blending may be performed in a Turbula blender or a v-shellblender. The components may be blended for 10 minutes without lubricantfollowed by additional blending with lubricant for 3 minutes.

Roller Compaction

The blend may be roller compacted in ribbons and milled into granulesusing an Alexanderwerk WP 120. The rolls used may be the 25 mm rollsusing a compaction pressure of 18 to 50 bar, a roller speed of 3 to 12RPM, and a screw feeder speed of 20 to 80 RPM. The screen sizes of theintegrated mill may be 2 mm for the top screen and 0.8 mm for the bottomscreen.

Blending

The roller compacted granules may be blended with extra-granularexcipients such as fillers and lubricant using a V-shell blender. Theblending time may be 5, 3 or 1 minute(s).

Compression

The compression blend has been compressed into tablets using a singlestation Riva MiniPress with 10 mm tooling. The weight of the tablets fora 100 mg dose may be about 200, 250, or 300 mg.

Film Coating

Tablets may be film coated using a pan coater, such as, for example anO′Hara Labcoat.

Printing

Film coated tablets may be printed with a monogram on one or both tabletfaces with, for example, a Hartnett Delta printer.

Tablet Formation from High Shear Granule Composition

Equipment/Process

Equipment

Granulator: Procept MiPro with a 250 ml or 1 L granulation bowl.

Screening/Weighing

Compound 1 and excipients may be screened prior to or after weigh-out.Possible screen sizes are mesh 20, mesh 40, or mesh 60. Compound 1 maybe pre-blended with one or more of the excipients to simplify screening.

Granulation Operation

Granulation Fluid—SLS and binder are added to purified water and mixeduntil dissolved. A suitable ratio is 2.5% w/w SLS and 10.0% w/w PVP K30in water.

Granulation—The excipients and compound 1 are added to the granulationbowl. The order of addition may be Compound 1, disintegrant, diluent,and filler. The components may be mixed in the 250 ml bowl for 1 minuteat impeller speed 1000 RPM and chopper speed 1000 RPM. Granulation maybe performed at an impeller speed of 2000 RPM with a chopper speed of4000 RPM while adding the granulation fluid with a syringe pump at 1.5to 4.5 g/min. The fluid addition time may be 4 to 12 minutes. After therequired binder fluid is added, the granules may be wet-massed for about10 seconds to about 1 minute. One notable advantage of the present highshear granulation process is using a granulation fluid that comprisesboth a surfactant and the binder for better granulation throughincreased wettability. In one embodiment, the surfactant is SLS.

Milling

The granules may be reduced in size using a screen mill or a cone mill.

Drying

The granules may be dried using a vacuum oven, tray dryer, bi-conicaldryer, or fluid bed drier. The granules have been dried using a vacuumoven with a nitrogen purge.

Blending

The granules may be blended with extra-granular excipients. The granuleshave been blended with extra-granular disintegrant, diluent, filler, andlubricant. The granules have been blended using the Turbula blender for3 minutes pre-lubricant and 1 minute with lubricant. A larger scaleblender such as a 4-quart V-shell blender may be used.

Compression

The compression blend has been compressed into tablets using a singlestation Riva MiniPress with 8 mm, or 10 mm tooling. The weight of thetablets for a 100 mg dose may be about 160, 200, or 250 mg.

Film Coating

Tablets may be film coated using a pan coater, such as, for example anO′Hara Labcoat.

Printing

Film coated tablets may be printed with a monogram on one or both tabletfaces with, for example, a Hartnett Delta printer.

Tablet Formation from Continuous Twin Screw Wet Granulation Process

Equipment/Process

Equipment

Granulator: ConsiGma or Leistritz or Thermo Fisher twin screwgranulator.

Screening/Weighing

Compound 1 and excipients may be screened prior to or after weigh-out.Possible screen sizes are mesh 20, mesh 40, or mesh 60. Compound 1 maybe pre-blended with one or more of the excipients to simplify screening.

Blending

Compound 1 and excipients may be added to the blender in differentorder. The blending may be performed in a Turbula blender, a v-shellblender, a bin blender, or a continuous blender. The components may beblended for 10 minutes for batch blenders or continuously for acontinuous blender.

Granulation Operation

Granulation Fluid—SLS and binder are added to purified water and mixeduntil dissolved. A suitable ratio is 2.5% w/w SLS and 10.0% w/w PVP K30in water.

Granulation—The blend containing Compound 1 and excipients may be dosedinto the twin screw granulator using a Loss in Weight feeder at a rateof 10 kg/hr. The granulation fluid may be added using a peristaltic pumpat a rate of 3.5 kg/hr. The granulator may be run at a speed of 400 RPM.A notable advantage of the present twin screw wet granulation process isusing a granulation fluid that comprises both a surfactant and thebinder for better granulation through increased wettability. In oneembodiment, the surfactant is SLS. Another notable advantage is thatbecause the process is continuous and at any moment in time only alimited amount of material is processed, the process can be wellcontrolled and results in a high quality product.

Milling

The granules may be reduced in size using a screen mill or a cone mill

Drying

The granules may be dried using a vacuum oven, tray dryer, bi-conicaldryer, or fluid bed drier.

Blending

The granules may be blended with extra-granular excipients. The granuleshave been blended using a 300 liter bin blender for 60 revolutions.

Compression

The compression blend has been compressed into tablets using a CourtoyModul P rotary press

Film Coating

Tablets may be film coated using a pan coater, such as, for example anO'Hara Labcoat.

Printing

Film coated tablets may be printed with a monogram on one or both tabletfaces with, for example, a Hartnett Delta printer.

Dosing Administration Schedule

In another aspect, the invention relates to a method of treating a CFTRmediated disease in a subject comprising administering to a subject inneed thereof an effective amount of the pharmaceutical compositionprovided by the invention. In another embodiment, the pharmaceuticalcomposition is administered to the subject once every two weeks. Inanother embodiment, the pharmaceutical composition is administered tothe subject once a week. In another embodiment, the pharmaceuticalcomposition is administered to the subject once every three days. Inanother embodiment, the pharmaceutical composition is administered tothe subject once a day. In one embodiment, when the pharmaceuticalcomposition is a tablet according to Table 9, 10, 11, 12, 13, 14, 15,16, 17, 18, or 19 dosing is once a day.

Assays

Assays for Detecting and Measuring F508del-CFTR Correction Properties ofCompounds

Membrane potential optical methods for assaying F508del-CFTR modulationproperties of compounds.

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

1. Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with F508del-CFTR; a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hrs at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hrs at 27° C. to “temperature-correct” F508del-CFTR. The cellswere subsequently rinsed 3× with Krebs Ringers solution and loaded withthe voltage-sensitive dyes. To activate F508del-CFTR, 10 μM forskolinand the CFTR potentiator, genistein (20 μM), were added along withCl⁻-free medium to each well. The addition of Cl⁻-free medium promotedCl⁻ efflux in response to F508del-CFTR activation and the resultingmembrane depolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

2. Identification of Potentiator Compounds

To identify potentiators of F508del-CFTR, a double-addition HTS assayformat was developed. During the first addition, a Cl⁻-free medium withor without test compound was added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2-10 μM forskolin was added toactivate F508del-CFTR. The extracellular Cl⁻ concentration followingboth additions was 28 mM, which promoted Cl⁻ efflux in response toF508del-CFTR activation and the resulting membrane depolarization wasoptically monitored using the FRET-based voltage-sensor dyes.

3. Solutions

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

Chloride-free bath solution: Chloride salts in Bath Solution #1 aresubstituted with gluconate salts.

CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored at −20°C. DiSBAC₂(3): Prepared as a 10 mM stock in DMSO and stored at −20° C.

4. Cell Culture

NIH3T3 mouse fibroblasts stably expressing F508del-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1× NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hrs at 37° C. before culturing at 27° C. for24 hrs for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16-24hours.

Electrophysiological Assays for Assaying F508del-CFTR ModulationProperties of Compounds

1. Ussing Chamber Assay

Using chamber experiments were performed on polarized epithelial cellsexpressing F508del-CFTR to further characterize the F508del-CFTRmodulators identified in the optical assays. FRT^(ΔF508-CFTR) epithelialcells grown on Costar Snapwell cell culture inserts were mounted in anUssing chamber (Physiologic Instruments, Inc., San Diego, Calif.), andthe monolayers were continuously short-circuited using a Voltage-clampSystem (Department of Bioengineering, University of Iowa, Iowa, and,Physiologic Instruments, Inc., San Diego, Calif.). Transepithelialresistance was measured by applying a 2-mV pulse. Under theseconditions, the FRT epithelia demonstrated resistances of 4 KΩ/cm² ormore. The solutions were maintained at 27° C. and bubbled with air. Theelectrode offset potential and fluid resistance were corrected using acell-free insert. Under these conditions, the current reflects the flowof Cl⁻ through F508del-CFTR expressed in the apical membrane. The Iscwas digitally acquired using an MP100A-CE interface and AcqKnowledgesoftware (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

2. Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate F508del-CFTR,forskolin (10 μM) and the PDE inhibitor, IBMX (100 μM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 μM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing F508del-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 μM of the test compound for24 hours at 37° C. and were subsequently washed 3× prior to recording.The cAMP- and genistein-mediated I_(SC) in compound-treated cells wasnormalized to the 27° C. and 37° C. controls and expressed as percentageactivity. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated Isc compared tothe 37° C. controls.

3. Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane and was permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large concentration gradientacross the epithelium. All experiments were performed 30 min afternystatin permeabilization. Forskolin (10 μM) and all test compounds wereadded to both sides of the cell culture inserts. The efficacy of theputative F508del-CFTR potentiators was compared to that of the knownpotentiator, genistein.

4. Solutions

Basolateral solution (in mM): NaCl (135), CaCl₂ (1.2), MgCl₂ (1.2),K₂HPO₄ (2.4), KHPO₄ (0.6),N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (10), anddextrose (10). The solution was titrated to pH 7.4 with NaOH.

Apical solution (in mM): Same as basolateral solution with NaCl replacedwith Na Gluconate (135).

5. Cell Culture

Fisher rat epithelial (FRT) cells expressing F508del-CFTR(FRT^(ΔF508-CFTR)) were used for Ussing chamber experiments for theputative F508del-CFTR modulators identified from our optical assays. Thecells were cultured on Costar Snapwell cell culture inserts and culturedfor five days at 37° C. and 5% CO₂ in Coon's modified Ham's F-12 mediumsupplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100μg/ml streptomycin. Prior to use for characterizing the potentiatoractivity of compounds, the cells were incubated at 27° C. for 16-48 hrsto correct for the F508del-CFTR. To determine the activity ofcorrections compounds, the cells were incubated at 27° C. or 37° C. withand without the compounds for 24 hours.

6. Whole-Cell Recordings

The macroscopic F508del-CFTR current (I_(ΔF508)) in temperature- andtest compound-corrected NIH3T3 cells stably expressing F508del-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5-6 MΩ when filled with the intracellular solution. Underthese recording conditions, the calculated reversal potential for Cl⁻(E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance >20 GΩ and a series resistance <15 MΩ. Pulse generation, dataacquisition, and analysis were performed using a PC equipped with aDigidata 1320 A/D interface in conjunction with Clampex 8 (AxonInstruments Inc.). The bath contained <250 μl of saline and wascontinuously perifused at a rate of 2 ml/min using a gravity-drivenperfusion system,

7. Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional F508del-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate F 508del-CFTR, 10 μM forskolin and 20 μM genisteinwere added to the cells. Under our recording conditions, the currentdensity following 24-hr incubation at 27° C. was higher than thatobserved following 24-hr incubation at 37° C. These results areconsistent with the known effects of low-temperature incubation on thedensity of F508del-CFTR in the plasma membrane. To determine the effectsof correction compounds on CFTR current density, the cells wereincubated with 10 μM of the test compound for 24 hours at 37° C. and thecurrent density was compared to the 27° C. and 37° C. controls (%activity). Prior to recording, the cells were washed 3× withextracellular recording medium to remove any remaining test compound.Preincubation with 10 μM of correction compounds significantly increasedthe cAMP- and genistein-dependent current compared to the 37° C.controls.

8. Identification of Potentiator Compounds

The ability of F508del-CFTR potentiators to increase the macroscopicF508del-CFTR Cl current (I_(ΔF508)) in NIH3T3 cells stably expressingF508del-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

9. Solutions

Intracellular solution (in mM): Cs-aspartate (90), CsCl (50), MgCl₂ (1),HEPES (10), and 240 μg/ml amphotericin-B (pH adjusted to 7.35 withCsOH).

Extracellular solution (in mM): N-methyl-D-glucamine (NMDG)-Cl (150),MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35 with HCl).

10. Cell Culture

NIH3T3 mouse fibroblasts stably expressing F508del-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1× NEAA, β-ME, 1× pen/strep, and25 mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

11. Single-Channel Recordings

The single-channel activities of temperature-corrected F508del-CFTRstably expressed in NIH3T3 cells and activities of potentiator compoundswere observed using excised inside-out membrane patch. Briefly,voltage-clamp recordings of single-channel activity were performed atroom temperature with an Axopatch 200B patch-clamp amplifier (AxonInstruments Inc.). All recordings were acquired at a sampling frequencyof 10 kHz and low-pass filtered at 400 Hz. Patch pipettes werefabricated from Corning Kovar Sealing #7052 glass (World PrecisionInstruments, Inc., Sarasota, Fla.) and had a resistance of 5-8 MΩ whenfilled with the extracellular solution. The F508del-CFTR was activatedafter excision, by adding 1 mM Mg-ATP, and 75 nM of the cAMP-dependentprotein kinase, catalytic subunit (PKA; Promega Corp. Madison, Wis.).After channel activity stabilized, the patch was perifused using agravity-driven microperfusion system. The inflow was placed adjacent tothe patch, resulting in complete solution exchange within 1-2 sec. Tomaintain F508del-CFTR activity during the rapid perifusion, thenonspecific phosphatase inhibitor F⁻ (10 mM NaF) was added to the bathsolution. Under these recording conditions, channel activity remainedconstant throughout the duration of the patch recording (up to 60 min).Currents produced by positive charge moving from the intra- toextracellular solutions (anions moving in the opposite direction) areshown as positive currents. The pipette potential (V_(p)) was maintainedat 80 mV.

Channel activity was analyzed from membrane patches containing ≤2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of F508del-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

12. Solutions

Extracellular solution (in mM): NMDG (150), aspartic acid (150), CaCl₂(5), MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with Tris base).

Intracellular solution (in mM): NMDG-Cl (150), MgCl₂ (2), EGTA (5), TES(10), and Tris base (14) (pH adjusted to 7.35 with HCl).

13. Cell Culture

NIH3T3 mouse fibroblasts stably expressing F508del-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1× NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Using the procedures described above, the activity, i.e., EC50s, ofCompound 1 has been measured and is shown in Table 20.

TABLE 20 IC50/EC50 Bins: +++ <= 2.0 < ++ <= 5.0 < + PercentActivityBins: + <= 25.0 < ++ <= 100.0 < +++ Cmpd. No. BinnedEC50BinnedMaxEfficacy 1 +++ +++

Other Embodiments

All publications and patents referred to in this disclosure areincorporated herein by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference. Should themeaning of the terms in any of the patents or publications incorporatedby reference conflict with the meaning of the terms used in thisdisclosure, the meaning of the terms in this disclosure are intended tobe controlling. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments of the invention. One skilled inthe art will readily recognize from such discussion and from theaccompanying drawings and claims, that various changes, modificationsand variations can be made therein without departing from the spirit andscope of the invention as defined in the following claims.

1-54. (canceled)
 55. A continuous process for preparing a tabletcomprising 3-(6-(1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid(Compound 1′) Form I, comprising the steps of: a) mixing Compound 1 FormI, a filler, and a disintegrant in a blender to form a blend; b)preparing a granulation solution with water, a binder, and a surfactant;c) feeding the blend from step a) into a continuous twin screwgranulator while adding the granulation solution from step b) to producegranules; d) drying the granules from step c) and milling them; e)blending the milled granules from step d) with a filler, disintegrant,and lubricant to form a blend; f) compressing the blend from step e)into a tablet; and g) optionally coating the tablet from step f),wherein the particle size of Compound 1 Form I is between 0.1 and 10microns.
 56. The process of claim 55, wherein the tablet comprises atleast 30 wt % by weight of Compound 1 Form I.
 57. The process of claim55, wherein the particle size of Compound 1 Form I is between 1 micronand 5 microns.
 58. The process of claim 55, wherein Compound 1 Form Ihas a particle size D50 of 2.0 microns.
 59. The process of claim 55,wherein the tablet has a target friability of less than 1.0% after 400revolutions.
 60. The process of claim 55, wherein the tablet has ahardness of at least 5 kP.
 61. The process of claim 55, wherein Compound1 Form I is characterized by one or more peaks within one or more 20ranges, selected from 15.2 to 15.6 degrees; 16.1 to 16.5 degrees; and14.3 to 14.7 degrees in an X-ray powder diffraction obtained using Cu Kalpha radiation.
 62. The process of claim 61, wherein Compound 1 Form Iis characterized by a peak within the range of 16.1 to 16.5 degrees inan X-ray powder diffraction obtained using Cu K alpha radiation.
 63. Theprocess of claim 62, wherein Compound 1 Form I is characterized by apeak having a 2θ value at 16.3 degrees in an X-ray powder diffraction.64. The process of claim 61, wherein Compound 1 Form I is characterizedby a peak within the range of 14.3 to 14.7 degrees in an X-ray powderdiffraction obtained using Cu K alpha radiation.
 65. The process ofclaim 64, wherein Compound 1 Form I is characterized by a peak having a2θ value at 14.5 degrees in an X-ray powder diffraction.
 66. The processof claim 61, wherein Compound 1 Form I is characterized by a peak withinthe range of 15.2 to 15.6 degrees in an X-ray powder diffractionobtained using Cu K alpha radiation.
 67. The process of claim 66,wherein Compound 1 Form I is characterized by a peak having a 2θ valueat 15.4 degrees in an X-ray powder diffraction.
 68. The process of claim61, wherein Compound 1 Form I is further characterized by a peak withinthe range of 17.6 to 18.0 degrees in an X-ray powder diffractionobtained using Cu K alpha radiation.
 69. The process of claim 61,wherein Compound 1 Form I is further characterized by a peak within therange of 7.6 to 8.0 degrees in an X-ray powder diffraction obtainedusing Cu K alpha radiation.
 70. The process of claim 55, whereinCompound 1 Form I is characterized by one or more peaks having a 2θvalue, ±0.2 degrees, selected from 14.41 degrees, 14.64 degrees, 15.23degrees, 16.11 degrees, 17.67 degrees, 19.32 degrees, 21.67 degrees,23.40 degrees, 23.99 degrees, 26.10 degrees, and 28.54 degrees, in anX-ray powder diffraction obtained using Cu K alpha radiation.
 71. Theprocess of claim 55, wherein Compound 1 Form I is characterized by oneor more peaks having a 2θ value, ±0.2 degrees, selected from 7.83degrees; 14.51 degrees; 14.78 degrees; 15.39 degrees; 16.26 degrees;16.62 degrees; 17.81 degrees; 21.59 degrees; 23.32 degrees; 24.93degrees; and 25.99 degrees, in an X-ray powder diffraction obtainedusing Cu K alpha radiation.
 72. The process of claim 55, whereinCompound 1 Form I is characterized by a diffraction patternsubstantially similar to that of FIG.
 1. 73. The process of claim 55,wherein Compound 1 Form I is characterized by a diffraction patternsubstantially similar to that of FIG.
 2. 74. The process of claim 55,wherein Compound 1 Form I is characterized as a monoclinic crystalsystem in P2₁/n space group with the following unit cell dimensions:a=4.9626(7) Å, b=12.299(2) Å, c=33.075 (4) Å, β=93.938(9)°.
 75. A tabletprepared by the process of claim
 55. 76. A method of treating cysticfibrosis in a patient comprising administering a tablet prepared by theprocess of claim
 55. 77. The method of claim 76, wherein the patient hasa ΔF508 CFTR mutation.
 78. The method of claim 77, wherein the patientis homozygous for the ΔF508 CFTR mutation.